CN1636030A - Reduced byproduct high solids polyamine-epihalohydrin compositions - Google Patents

Abstract

Processes for rendering a polyamine-epihalohydrin resin storage stable, including processes that preparate a storage stable resing and/or process that treat resins. A composition containing a polyamine-epihalohydrin resin with a solids content of at least 15 wt% treated with at least one enzymatic agent. A process for treating polyamine-epihalohydrin resin, to reduce the level of the nitrogen-free organohalogen compound by adding at least one microorganism, or at least one enzyme isolated from the at least one microorganism to an aqueous composition containing a solids content of least 15 wt%, under conditions to dehalogenate the nitrogen-free organohalogen compound so as to reduce a level of the nitrogen-free organohalogen compound while leaving the polyamine-epihalohydrin resin substantially intact. A paper product containing the storage stable polyamine-epihalohydrin resin, when corrected for adding at about a 1 wt% addition level of the resin, contains less the about 250 ppb of CDP.

Description

High solids polyamine-epihalohydrin compositions with reduced by-products

Field of Invention

The present invention relates to resins and aqueous resin-containing compositions, and methods of forming resin compositions, particularly for use in the paper industry, including strength agents, such as wet and dry strength agents, and creping agents. The present invention also relates to resins and methods for their production, wherein the resins, and compositions and products containing these resins, such as paper products, have reduced residues, such as epihalohydrins and epihalohydrin hydrolysates. Additionally, the present invention relates to resins, and compositions and products, such as paper products, that retain low levels of residues, such as epihalohydrins and epihalohydrin hydrolysates, when stored. Furthermore, each aspect of the invention relates to compositions having resins of different solids content, especially high solids content.

Background of the Invention

Wet strength resins are often added to paper and board during manufacture. In the absence of wet strength resins, paper normally retains only 3%-5% of its strength after wetting with water. However, papers made with wet strength resins typically retain at least 10% to 50% of their strength when wet. Wet strength is used in a variety of paper applications, some examples of which are toweling, milk and juice cartons, paper bags and liners for corrugated containers.

Dry strength is also a key paper property, especially in light of the recent trend of papermakers to use high yields of wood pulp in paper for lower costs. These high yield wood pulps generally produce papers of significantly lower strength when compared to paper produced from highly refined pulps.

Commercially available wet strength resins include Kymene(R) 557H, Kymene(R) 557LX, Kymene(R) SLX, Kymene(R) Plus, Kymene(R) 450, and Kymene(R) 736 wet strength resins, available from Hercules Incorporated, Wilmington, Del. Wet strength resins, such as those listed above, also provide increased dry strength to the paper.

Resins similar to those used to impart strength to paper are also often used as creping binders. In the production of some paper products, such as facial tissue, toilet tissue or toweling, the paper web is often creped to impart desired textural characteristics, such as softness and bulk. Creping typically involves bonding the web, in the case of paper, a cellulosic web, to a rotating creping cylinder, such as a device known as a Yankee dryer, and then using a doctor blade to cause the bonded Nets separate. The impact of the web against the blade breaks some of the fiber-fiber bonds in the web and wrinkles or puckers the web.

The extent of this creping action depends on many factors, including the degree of adhesion between the wire and the surface of the creping cylinder. Greater tack results in increased softness, albeit usually at a loss of some strength. To increase tack due to the moisture content in the web, any naturally occurring tack that the web may have can be enhanced with a creping adhesive, which will vary widely depending on the degree to which the web was previously dried. The creping adhesive should also prevent abrasion of the dryer surface and provide lubrication between the blade and dryer surface and reduce chemical attack, as well as control the degree of wrinkling. A creping adhesive coating that makes the sheet just strong enough to bond to the drum will impart good creases, imparting absorbency and softness with minimal loss of paper strength as possible. If the adhesion to the dryer drum is too strong, the flakes may pick up or even "plug", ie move under the blade, and wrap around the dryer drum. Without enough tack, the sheet will take off too easily and wrinkle too little.

Creping adhesives, usually in aqueous solution or dispersion, are usually sprayed onto the surface of a creping drum or drum, such as a Yankee dryer. This promotes heat transfer, allowing the flakes to dry more efficiently. If the pulp equipment adheres too strongly to the creping cylinder, a release agent can be sprayed onto the cylinder. The release agent is typically a hydrocarbon oil. These agents help release the web evenly on the creping blade and also lubricate and prevent excessive blade wear.

Examples of creping adhesive compositions include those disclosed in US 5,187,219 to Furman (incorporated herein by reference in its entirety). These compositions contain a water-soluble glyoxylated acrylamide/diallyldimethylammonium chloride polymer and a water-soluble polyol having a molecular weight below 3000 as a plasticizer for the polymer.

US 5,246,544 to Hollenberg et al. (incorporated herein by reference in its entirety) discloses a reversibly crosslinked creping adhesive comprising non-self-crosslinking materials, such as polymers having functional groups that crosslink by ionically crosslinking or oligomers and at least one metal, tetravalent or higher cationic crosslinker. Contains additives to improve the mechanical properties of cross-linked polymers, such as diols, polyethylene glycols and other polyols such as mono- and oligosaccharides.

Polyaminoamide/epichlorohydrin creping adhesives are disclosed in US 5,338,807 to Espy et al, US 5,994,449 to Maslanka, and Canadian Patent 979,579 to Giles et al (which are incorporated herein by reference in their entirety).

US 5,374,334 to Sommese et al., which is incorporated herein by reference in its entirety, discloses a creping adhesive which is a crosslinked vinylamine/vinyl alcohol polymer containing from about 1 to about 99% vinylamine. Epichlorohydrin is disclosed as a crosslinking agent.

US 4,684,439 and 4,788,243 to Soerens, which are incorporated herein by reference in their entirety, disclose creping adhesives comprising a mixture of polyvinyl alcohol and a water-soluble thermoplastic polyamide resin comprising polyalkylene polyamine, saturated fatty acid The reaction product of a family of dicarboxylic acids and poly(ethylene oxide) diamines.

In US 4,501,640 and 4,528,316 to Soerens, which are incorporated herein by reference in their entirety, a creping adhesive is disclosed comprising a mixture of polyvinyl alcohol and a water-soluble, thermosetting cationic polyamide resin.

Commercially available creping adhesives include Crepetrol(R) 190, Crepetrol(R) 290, and Crepetrol(R) 80E cationic polymers, available from Hercules Incorporated, Wilmington, Del.

Furthermore, polyamine-epihalohydrin resins, such as polyaminopolyamide-epihalohydrin resins, often contain significant amounts of epihalohydrin hydrolysis products. For example, commercial polyaminopolyamide-containing chloropropane resins typically contain 1-10% by weight dry basis of epichlorohydrin (epi) by-products, 1,3-dichloropropanol (1,3-DCP), 2, 3-dichloropropanol (2,3-DCP) and 3-chloropropanediol (CPD). The epi by-products are also known as epi residues. The production of these resins with reduced amounts of epi by-products has been the subject of much research. There is increasing environmental pressure to produce resins with lower amounts of adsorbable organic halogen (AOX) species. "AOX" refers to the adsorbable organic halogen content of the resin, which can be determined by adsorption on carbon. AOX includes epichlorohydrin (epi) and epi by-products (1,3-dichloropropanol, 2,3-dichloropropanol, and 3-chloropropanediol) and organic halides attached to the polymer backbone.

Several methods have been devised to reduce the amount of epihalohydrin hydrolysis products. Reducing the amount of epihalohydrin used in the synthetic steps is additionally taught in US5171795. The result is a longer reaction time. In US5017642 it is taught to control the production process to produce compositions of reduced concentration of hydrolyzate. The entire contents of these patents are incorporated herein by reference.

Post-synthesis processing is also taught. US5256727, which is hereby incorporated by reference in its entirety, teaches the reaction of epihalohydrins and their hydrolysates with dibasic phosphates or alkanolamines in equimolar ratios to convert chlorinated organic compounds to non-chlorinated species. For this purpose the second reaction step must be carried out for at least 3 hours, which significantly increases costs and produces large amounts of unwanted organic or inorganic materials in the wet strength composition. In compositions containing substantial amounts of epihalohydrin and epihalohydrin hydrolyzate (eg, about 1-6% by weight of the composition), the amount of organics formed is also present in undesirably large amounts.

US 5,516,885 and WO 92/22601, which are incorporated herein by reference in their entirety, include the use of ion exchange resins to remove halogenated by-products from products containing large amounts of halogenated by-products and low amounts of halogenated by-products. However, it is clear from the presented data that the yield of the wet strength composition is significantly reduced and the wet strength efficacy is reduced.

It is known that compounds containing nitrogen-free organohalogens can be converted into relatively harmless substances. For example, 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol (also known as 3-chloropropanediol, 3-monochloropropanediol, monochloropropanediol, chloropropanediol, CPD, 3- CPD, MCPD and 3-MCPD) and epichlorohydrin to produce glycerol with alkali.

It is also known to convert nitrogen-free organohalogen compounds with microorganisms containing dehalogenases. For example, CECastro et al. ("Biological Cleavage of Carbon-Halogen Bonds Metabolism of 3-Bromopropanol by Pseudomonas sp.", Biochimica et Biophysica Acta , 100 , 384-392, 1962) (which is incorporated herein by reference in its entirety) describe Use of an isolated Pseudomonas species for the sequential metabolism of 3-bromopropanol to 3-bromopropionic acid, 3-hydroxypropionic acid, and _CO2 .

A number of US patents also describe the use of microorganisms for the dehalogenation of halohydrins, eg US4452894, 4477570 and 4493895. Each of these patents is incorporated herein by reference as if fully set forth herein.

US5470742, 5843763 and 5871616 (which are incorporated herein by reference in their entirety) disclose the use of microorganisms or enzymes derived from microorganisms for the removal of epihalohydrins and epihalohydrins from wet strength compositions without reducing wet strength efficacy Use of hydrolyzate.

US application 09/629629, filed July 31, 2000, which is hereby incorporated by reference in its entirety, relates to the use of microorganisms or enzymes derived from microorganisms for the removal of epihalohydrins and epihalohydrin hydrolysates from resin compositions , and discloses a preferred continuous method for the growth of these microorganisms.

Furthermore, US5972691 and WO 96/40967 (which are incorporated herein by reference in their entirety) disclose that it has been achieved to treat the wet strength composition with an inorganic base after the synthesis step (i.e. after the polymerization reaction to form the resin) and to convert the resin at low pH. Stabilizing, thereby reducing the organic halide (eg, chlorinated hydrolyzate) content of the wet strength composition to a suitable amount (eg, about 0.5% by weight of the composition). The composition so formed can then be treated with microorganisms or enzymes to economically produce a wet strength composition with very low levels of epihalohydrin and epihalohydrin hydrolyzate.

It is also known that epihalohydrins and epihalohydrin hydrolysates can react with bases to form chloride ions and polyols. US4975499 teaches the use of bases during synthesis steps to reduce the organochlorine content of wet strength compositions to suitable amounts (eg, to suitable amounts of from about 0.11 to about 0.16% by weight of the composition). US5019606 teaches reacting wet strength compositions with organic or inorganic bases. The entire contents of these patents are incorporated herein by reference.

Moreover, US application 09/001787, filed December 31, 1997, and 09/224107 and WO 99/33901, filed December 22, 1998 by Riehle, which are incorporated herein by reference in their entirety, disclose, among other features, , a method of reducing the AOX content of a raw water-soluble wet-strength resin containing azetidinium ions and tertiary aminohalohydrins, comprising treating an aqueous resin solution with a base to form a treated resin wherein at least about 20 mol% of the tertiary aminohalohydrin was converted to epoxide with essentially no change in the amount of azetidinium ion, and the treated resin was at least as effective at imparting wet strength as the raw wet strength resin.

Also, US Patent Applications 09/592681, filed June 12, 2000, 09/363224, filed July 30, 1999, 09/330200, filed June 11, 1999 (incorporated by reference in their entireties) It relates to polyamine-epihalohydrin resin products, in particular polyamine-epihalohydrin resin products which can be stored with at least reduced formation of halogen-containing residues such as 3-chloropropanediol (CPD). Furthermore, these applications disclose the use of microorganisms or enzymes derived from microorganisms to remove epihalohydrins and epihalohydrin hydrolyzates from wet strength compositions without loss of wet strength efficacy.

WO 99/09252 describes thermosetting wet strength resins prepared from end-capped polyaminoamide polymers. The cappers used are monocarboxylic acids or monofunctional carboxylic acid esters and are used to control the molecular weight of the polyaminoamides in order to obtain wet strength resins with high solids content.

Each of the preceding approaches has provided varying results, and there is a continuing need for improvement in the use of polyamine-epihalohydrins, especially at high solids levels. In particular, there remains a need for resin compositions, such as wet strength, dry strength and creping agent resins, which can be provided as solutions or dispersions at reasonable viscosities relative to high polymer solids content. Accordingly, there remains a need for resins that are prepared, stored, handled and shipped as dispersions or solutions containing high solids concentrations, and which do not have polymer crosslinked product deterioration, such as gelation problems.

Summary of Invention

Enzyme treatment of tertiary amine-based resins can be carried out at the higher concentrations previously disclosed when properly balanced conditions of time, temperature, pH and enzyme concentration are used.

The present invention relates to polyamine-epihalohydrin resin products, in particular polyamine-epihalohydrin resin products which can be stored with at least reduced formation of halogen-containing residues such as 3-chloropropanediol (CPD).

The present invention also relates to the different uses of polyamine-epihalohydrin resins having at least reduced formation of halogen-containing residues, for example as strength agents, including wet and dry strength agents, and creping agents.

The present invention also relates to polyamine-epihalohydrin resin products, especially paper products, having reduced CPD formation on storage.

The present invention also relates to various treatments of polyamine-epihalohydrin resins, including treatments to reduce the concentration of halogen-containing residues associated with the resins and/or compositions containing these resins.

The present invention also relates to the preparation of storage-stable polyamine-epihalohydrins and/or the treatment of polyamine-epihalohydrin resins to render these resins storage-stable, especially at high solids content.

Detailed description of the invention

All percentages, parts, ratios, etc. are by weight unless otherwise indicated.

Unless otherwise stated, the compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as a mixture of compounds.

Moreover, when an amount, concentration or other value or parameter is given as a list of preferred values above and preferred values below, this is to be understood as specifically disclosing all ranges formed by any pair of preferred values above and values below, Regardless of whether the scope is disclosed separately.

US patent applications 09/592,681, filed June 12, 2000, 09/363,224, filed July 30, 1999, and 09/330,200, filed June 11, 1999 (which are hereby incorporated by reference in their entireties) relate to the discovery The formation of CPD in polyamine-epihalohydrin resins after storage was observed to be due to the association of the CPD species formed with the oligomeric and/or polymeric components of the resin. It is disclosed in these applications that polyamine-epihalohydrin resins can be treated in such a way during and/or after production in order to prevent the formation, inhibition and /or remove these elements. For example, these applications disclose acid treatment, base treatment, reduction of acid end groups in prepolymers, and enzyme treatment to remove or reduce CPD forming species.

Thus, in one aspect of the invention disclosed in US Patent Application 09/592681, polyamine-epihalohydrins with reduced CPD formation on storage and minimized levels of CPD in paper products can be produced by treating the resin with an enzymatic reagent. Resin products. Thus, CPD-forming species can be reduced and/or removed by treating the resin with an enzymatic reagent capable of releasing the CPD-forming species from the resin. The enzymatic reagent may include one or more enzymes capable of releasing CPD-forming species from the resin, such as at least one of esterases, lipases and proteases. Preferably the enzyme reagent has esterase activity. It is known to those skilled in the art that protease-type enzymes can have esterase activity and that esterase-type enzymes can have protease activity. Preferred proteases are the subtilisin group (E.C.3.4.31.62. Homology modeling and protein engineering strategy of subtilases. the family of subtilisin-like serineproteinases, Siezen RJ, de Vos WM, Leunissen JA, Dijkstra BW, Protein Eng 4, 1991, 719-37), especially enzymes produced by Bacillus licheniformis (Swiss-Prot AccessionNumber: P00780), Bacillus amyloliquefaciens (P00782) and Bacillus lentus (P29600). The enzyme may be in pure form, or the enzyme may not be purified. Also, mixtures of enzymes may be used, which mixtures may include mixtures of pure enzymes, mixtures of unpurified enzymes, or mixtures of both. In particular, the preferred enzyme reagents are ALCALASE and SAVINASE, both of which are available from Novozymes North America, Inc. Franklinton, North Carolina (formerly known as Novo Nordisk Biochem, North America, Inc.).

Extending from the above, in previous work polyamine-epihalohydrin resins having about 12-13.5 wt% solids were treated with ALCALASE 2.5L type DX (Novozymes) to reduce or remove CPD forming species. Under these processing conditions, eg pH 8, 40°C, 6-8 hours and 30 g of resin with 0.25 g of ALCALASE, the resin tends to exhibit high viscosity and become unusable. It has been surprisingly found according to the present invention that by balancing the processing conditions, including pH, temperature, concentration of enzyme reagents, initial viscosity and solids concentration of compositions containing polyamine-epihalohydrin resins, e.g. polyaminopolyamide-epihalohydrin Alcohol resin compositions that can be treated with enzymatic agents to reduce or remove CPD forming species, and that have desirable viscosity characteristics and exhibit excellent CPD release. These newly discovered enzyme treatment conditions increase, decrease or maintain resin viscosity at desired levels and allow enzyme treatment at low solids, as well as high solids of 15 wt% or greater.

Without wishing to be bound by theory, it is believed that as the active solids content increases, the rate of crosslinking increases and thus the viscosity increases. By proper choice of reaction conditions, the rate of the viscosity-increasing cross-linking reaction can be balanced with the rate of the enzymatic hydrolysis reaction, which lowers the viscosity so that the desired viscosity can be predictably obtained.

The present invention is useful because it enables higher yield enzymatic production and because lower levels of expensive enzymes can be used. The present technology thus enables (1) the production of high solids, high potency resins by longer azetidinium formation and (2) the production of resins containing lower AOX resin.

Thus, according to the present invention, it has been found that enzyme treatments to reduce or remove CPD forming species can be carried out at higher solids levels than desired. In this regard, the enzymatic treatment example in the aforementioned US Patent Application 09/592681 was performed at about 13-14 wt% solids. Thus, the enzyme treatment of the present invention may include solids levels disclosed in the prior art, including concentrations as low as 4 wt% or less. However, contrary to the prior art, the solids content of aqueous resin compositions treated with enzyme agents according to the invention may be higher than 15 wt%, more preferably higher than about 20 wt%, and especially together with creping agents may be higher than about 25 wt%. %. Preferred solids content ranges include about 15-50 wt%, more preferably about 18-40 wt%. For wet strength agents, the preferred solids content is about 15-40 wt%, more preferably about 18-25 wt%, a preferred solids value is about 21 wt%; and, for creping agents, the solids content is about 20-25 wt%. 40 wt%, more preferably about 22-30 wt%, a preferred solids value is about 26 wt%.

The terms "creping aid, creping resin, creping agent and creping adhesive" are used interchangeably and have the same meaning throughout the specification.

At least one enzymatic reagent is added to the resin under suitable conditions to achieve sufficient hydrolysis of the CPD forming species in the high resin solids composition. Preferably, the conditions of time, temperature, pH, enzyme concentration, onset viscosity, and solids content are balanced to enable the hydrolysis reaction while minimizing reduction in resin properties, such as resin wet strength or creping effectiveness, or preventing undesirably high resin viscosity . Thus, by balancing the conditions of time, temperature, pH, enzyme concentration, onset viscosity and solids content, the hydrolysis of CPD forming species can surprisingly be performed at high solids concentrations. For example, as the solids concentration increases, the pH and/or temperature should often decrease. Also, as the solids concentration increases, the enzyme concentration should often be increased.

Note that the viscosity of the resin composition may increase or decrease from the initial viscosity during enzyme treatment, and may remain the same or substantially the same, depending on the reaction conditions as described above. In the case of creping agents, it is often preferred, but not limited to, that the viscosity at the end of the enzyme treatment be the same or substantially the same as the starting viscosity. For example, in the case of wet strength agents, it is often preferred, but not limited to, to maintain or decrease from the initial viscosity at the beginning of the processing time and then maintain or increase to the desired viscosity at the end of the processing time. For example, for a resin having an initial Brookfield viscosity of about 100-300 cps and about 20-22 wt% active solids, conditions are preferably selected so that after processing, the resin viscosity is maintained or decreased with about 19-22 wt% active solids. And for example, for a resin having an initial Brookfield viscosity of about 100-300 cps and about 20-22 wt% active solids, it is preferred that the Gardner Holdt viscosity at the start of the reaction be about Approx. F at the end. Also for example, for a resin having an initial Brookfield viscosity of about 100-300 cps and about 20-22 wt% active solids, it is also preferred if the Gardner-Holdt viscosity at the start of the reaction is about G-J, ideally the Gardner-Holdt viscosity is While decreasing to about A-E at the end of the reaction, it is desirable to increase the process temperature until the Gardner-Holdt viscosity increases to about F-I. Also for example, for Kymene® E7219 (available from Hercules Incorporated, Wilmington, DE) with an initial Brookfield viscosity of about 200-300 cps and about 20-22 wt% active solids, if the Gardner-Holdt viscosity at the start of the reaction is about 1 , then ideally the Gardner-Holdt viscosity decreases during the reaction to about F at the end of the reaction so that the final resin (stabilized at about pH 3-3.5) has a Brookfield viscosity of about 100-150 cps. For example, for Kymene® E7219 (available from Hercules Incorporated, Wilmington, DE) with an initial Brookfield viscosity of about 200-300 cps and about 20-22 wt% active solids, if the Gardner-Holdt viscosity at the start of the reaction is about 1, It is then desirable that the Gardner-Holdt viscosity decrease during the reaction to about C at the end of the reaction, ideally the process temperature is increased until the Gardner-Holdt viscosity increases to about F.

For example, in the case of creping agents, it is often preferred, but not limited to, to have an initial viscosity of less than about 150 cps, more preferably less than about 100 cps, more preferably less than about 80 cps, and even more preferably less than about 40 cps. Preferably the initial viscosity of the reaction mixture is in the range of about 10 cps to 150 cps, more preferably about 20 cps to 100 cps, even more preferably about 40 to 80 cps.

With regard to the above, it is preferred that side reactions, such as polymerization failure or molecular weight increase, be minimized or at least balanced so that the viscosity of the reaction mixture remains below that which does not allow the reaction to proceed. Preferably, viscosity is measured using a Brookfield LVDV-II+ Programmable Viscometer at 25°C, or an equivalent such as a Brookfield DV II+, Spindle LV2 at 60 or 100 rpm, depending on its viscosity. For programmable viscometers, the procedure used is based on Operating Instructions, Manual No. M/97-164. The viscometer will measure viscosity only if the correct spindle and rpm are used for the viscosity of the sample according to the instruction manual.

Preferably the properties of the creping agent are almost the same after treatment as before treatment. Thus, as mentioned above, it is preferred to keep the viscosity of the reaction mixture constant or substantially constant during the reaction of the creping agent. Specifically, the viscosity of the reaction mixture does not increase by more than about 50%, more preferably not more than about 20%, and most preferably not more than about 10% of the starting viscosity.

It should also be noted that conditions, preferably temperature, pH and concentration of enzyme reagents, can be varied during the reaction. For example, if the concentration of the reaction mixture is higher than desired, the pH and/or temperature can be lowered and/or other enzymatic reagents can be added. Conversely, the pH and/or temperature can be increased, for example, if the viscosity of the reaction mixture is lower than desired.

The present invention also relates to a method of reducing the molecular weight or viscosity of a polyamine-epihalohydrin resin-containing composition comprising treating the polyamine-epihalohydrin resin-containing composition with at least one enzymatic agent. The composition may contain a high solids content, for example a solids content of at least 15 wt%. Changes in reaction conditions often alter reaction times. Its pH and/or temperature can be lowered and/or other enzymatic reagents can be added.

Specific examples of preferred conditions for wet strength resins and using ALCALASE 2.5L Type DX as the enzyme include the following. For resins having an initial Brookfield viscosity of about 150-300 cps and about 20-22 wt% active solids, it is preferred to use a temperature of about 20-33°C, a pH of about 6.8-7.8, ALCALASE 2.5L Type DX (used as is) and active The ratio of solids is about 1.0:20-1.0:5.0. More particularly, for Kymene® E7219 (available from Hercules Incorporated, Wilmington, DE) having an initial Brookfield viscosity of about 200-300 cps and about 20-22 wt% active solids, it is preferred to use a temperature of about 23-27°C, about At a pH of 6.8-7.5, the ratio of ALCALASE 2.5L Form DX (used as received) to active solids is about 1.0:8.0-1.0:18.0, and the treatment time is 6-10 hours. It should be noted that the amount of CPD released from the CPD-generating material desirably increases as the treatment time increases, with a preferred treatment time being 6-10 hours. Another example of conditions is as follows: for Kymene® E7219 (available from Hercules Incorporated, Wilmington, DE) with an initial Brookfield viscosity of about 200-300 cps and about 20-22 wt% active solids, the temperature is about 35°C and the pH is about 7.5, ALCALASE 2.5L Form DX (used as is) has a weight to active solids ratio (by weight) of approximately 1.0:8.3.

The temperature may be at least about 0°C, more preferably about 10°C-80°C, even more preferably about 20-60°C, more preferably about 20°C-40°C, more preferably about 20°C-30°C. The reaction time may be about 3 minutes to 350 hours, more preferably about 30 minutes to 48 hours, more preferably about 30 minutes to 96 hours, more preferably about 1 hour to 24 hours, even more preferably about 2 hours to 12 hours. The pH of the enzyme treatment will depend on the pH dependence of the particular enzyme and other treatment conditions, and can vary between 1-11, preferably 2-10, even more preferably about 2.5-9, even more preferably about 7-9, even More preferably 7-8. Other preferred pH ranges include: 5.0-8.0, 5.5-7.5, 6-9, 6-8.5, 6.5-8.

For example, the combination treatment can be started at pH 6.8-7.8 for 4-24 hours before and lowered to pH 5.5-7.0, or the pH shifted to 6.5-7.2 for 8-48 hours after the combination treatment.

The enzyme concentration will depend on its activity. For example, without limitation, the enzyme may be present in the following amount: about 0.04 g active enzyme dry basis per 1600 g polyamine-epichlorohydrin resin dry basis - 0.04 g active enzyme dry basis per 1.5 g polyamine-epichlorohydrin resin dry basis Base, the enzyme may also be present in the following amount: about 0.04 g active enzyme dry basis per 160 g polyamine-epichlorohydrin resin dry basis - 0.04 g active enzyme dry basis per 4 g polyamine-epichlorohydrin resin dry basis.

The enzyme concentration will depend on its activity. For example, but not limited to, when ALCALASE, the enzyme may be present in the following amount: about 1 g of ALCALASE Type 2.5L DX (used as is)/1600 g polyamine-epichlorohydrin resin dry basis - 1 g of ALCALASE Type 2.5L DX ( As-is used)/1.5g polyamine-epichlorohydrin resin dry base, enzyme may also be present in the following amount: About 1g of ALCALASE 2.5L type DX (as-is used)/160g polyamine-epichlorohydrin resin dry base-1g ALCALASE 2.5L Type DX (used as is)/4g polyamine-epichlorohydrin resin dry base.

Note that, following the guidelines and non-limiting examples set forth in this application, one of ordinary skill in the art will be able to determine the processing conditions and balance of processing conditions to achieve hydrolysis of CPD forming species at high solids concentrations and/or to achieve molecular weight or reduction in viscosity. For example, as the concentration of solids increases, its pH and/or temperature should generally be lowered, and the concentration of enzyme reagents often increased. Also, following these guidelines, one of ordinary skill in the art will be able to identify enzymatic reagents for removing CPD forming species and/or achieving molecular weight or viscosity reduction.

Furthermore, the preferred reaction conditions can be varied by using the appropriate type and amount of enzyme. For example, if the enzyme reagent has higher protease activity than esterase activity (protease/esterase balance) for polyamine-epichlorohydrin resins, then the reaction conditions can be changed to higher pH, temperature and/or solids, For example, the reaction conditions are above about pH 8 and/or the temperature is above about 40°C and/or the solids are above about 40 wt%. The practical definition is to obtain a resin with reduced CPD formation while having the desired viscosity. Although the conditions depend on the balance of esterase and protease activities of the particular enzyme, the preferred conditions of the present invention for ALCALASE 2.5L type DX are as follows: 15-50 wt% active solids, pH 6.9-7.9, at 0-35°C , for 4-24 hours and 8-20g active solids against 1g of ALCALASE 2.5L Type DX (used as is), and starting viscosity is 10cP-1000cP. Also, it should be noted that throughout this application the term "enzyme reagent concentration" is used. However, those of ordinary skill in the art will understand that enzymes may have different activities, and the concentration of enzymes may be adjusted according to their activities.

This enzymatic treatment can be used on resins produced in resin synthesis without further treatment. Furthermore, the resin can be treated by different methods prior to reducing and/or removing the CPD forming species. Furthermore, after the treatment to reduce and/or remove the CPD forming species, the resin can be treated by different methods. Furthermore, the resin can be treated differently before the reduction and/or removal of the CPD forming species, and the resin can be treated differently after the reduction and/or removal of the CPD forming species. For the sake of brevity, a full description of these methods is not repeated here, and reference is made to the above-cited US patent applications 09/629629, 09/592681, 09/363224, and 09/330200 (which are incorporated herein by reference in their entirety).

The resins of the present invention can be stored without excessive CPD formation. More specifically, for example, the solution will contain less than about 10 ppm (parts per million), more preferably less than about ppm, most preferably less than 1 ppm CPD when stored at about 13.5 wt% resin solids. In the context of the present invention, the phrase "resin solids" means the reactive polyamine-epihalohydrin in the composition.

In order to determine the storage stability of the resin solutions of the present invention, a resin solution stability test was carried out in which the resin solution was stored for 2 weeks at 50°C and a pH of about 2.5-8, preferably 2.8, and its CPD was determined at the end of the 2 week period content. Thus, if it is determined to contain less than about 250 ppm dry basis of CPD at the end of the 2 week period, more preferably less than about 150 ppm dry basis of CPD at the end of the 2 week period, more preferably less than about 150 ppm dry basis at the end of the 2 week period A CPD of about 75 ppm dry basis, even more preferably less than about 40 ppm dry basis of CPD measured at the end of the 2 week period, even more preferably less than about 10 ppm dry basis of CPD measured at the end of the 2 week period, then the present invention contains Solutions of amine-epihalohydrins will be storage stable.

Resin solution stability testing can be performed on solutions containing various percentages of resin solids; however, the resulting CPD should be calibrated against the solids content. For example, for a 15 wt % resin solids solution with a measured CPD content of 15 ppm, the calibrated CPD, on a dry basis, would be 100 ppm dry basis (15 ppm/0.15 wt resin solids).

Resin solution stability tests were performed by pouring a portion of the polyamine-epihalohydrin resin into a vessel with a stirrer. The vessel was placed in a 50°C water bath and maintained at 50°C with stirring. An aliquot was removed from the container and analyzed by GC (Gas Chromatography) following the GC procedure described below. Typically, the sample is first analyzed using a flame ionization detector (FID). Electrolytic conductivity detectors (ELCDs) or halogen specific detectors (XSDs) are used when increased sensitivity is required, especially when the species to be analyzed is below about 20 ppm. Other sensitive detectors can be used, such as electron capture detectors. This test is an accelerated aging test for a model aging at about 32°C for an extended period of time.

A further test for determining the storage stability of resin solutions according to the invention is the following test ("acid test"): A portion of the resin to be tested is added to a vessel containing a stirrer. Its pH was adjusted to pH 1.0 with 96 wt% sulfuric acid. The vessel was sealed and placed in a 50°C water bath and maintained at 50°C with stirring. An aliquot was removed from the container at 24 hours and analyzed by GC in the manner described below to provide an indication of storage stability.

This acid test can be performed on solutions containing various percent resin solids; however, the resulting CPD should be calibrated against solids. For example, for a 15 wt % resin solids solution with a measured CPD content of 15 ppm, the calibrated CPD, on a dry basis, would be 100 ppm dry basis (15 ppm/0.15 wt resin solids).

For embodiments of the invention where enzyme treatment is used for resins that do not require further treatment in the resin synthesis process, although further treatment can be used, the amount of CPD released and/or produced by the resin, when stored at pH 1, 50°C 24 hours and when measured in 24 hours, release and/or produce the CPD of less than about 1000ppm dry basis, more preferably release and/or produce the CPD of less than about 750ppm dry basis, even more preferably release and/or produce the CPD of less than about 750ppm dry basis 500ppm dry basis of CPD, even more preferably release and/or produce less than about 250ppm dry basis of CPD, even more preferably release and/or produce less than about 200ppm dry basis of CPD, even more preferably release and/or produce less than about 200ppm dry basis of CPD CPD of about 150 ppm dry basis, even more preferably release and/or produce CPD of less than about 100 ppm dry basis, even more preferably release and/or produce CPD of less than about 75 ppm dry basis, even more preferably release and/or produce CPD of less than about 75 ppm dry basis CPD at about 50 ppm dry basis, even more preferably release and/or produce CPD below about 25 ppm dry basis, even more preferably release and/or produce CPD below about 15 ppm dry basis, even more preferably release and/or produce CPD on dry basis Less than about 5 ppm dry basis of CPD, even more preferably less than about 3 ppm dry basis of CPD released and/or produced, even more preferably less than about 1 ppm dry basis of CPD released and/or produced.

For embodiments of the invention where the enzyme treatment is concurrent with, before, or after an additional treatment to reduce one of epihalohydrins, epihalohydrin by-products, and organohalogens attached to the polymer backbone, the The additional treatment may be, but is not limited to, combining the reduced CPD-forming resin with at least one microorganism, or separating the At least one enzyme contacted, when stored at pH 1, 50°C for 24 hours and measured at 24 hours, contains less than about 1000 ppm dry basis of CPD, more preferably contains less than about 750 ppm dry basis of CPD, even more preferably contains CPD less than about 500 ppm dry basis, even more preferably less than about 250 ppm dry basis CPD, even more preferably less than about 200 ppm dry basis CPD, even more preferably less than about 150 ppm dry basis CPD, even more Preferably less than about 100 ppm dry basis of CPD, even more preferably less than about 75 ppm dry basis of CPD, even more preferably less than about 50 ppm dry basis of CPD, even more preferably less than about 25 ppm dry basis of CPD, Even more preferably CPD containing less than about 15 ppm dry basis, even more preferably CPD containing less than about 5 ppm dry basis, even more preferably CPD containing less than about 3 ppm dry basis, even more preferably CPD containing less than about 1 ppm dry basis CPD.

GC Procedure and Instrumentation: Epi and epi by-products in treated and untreated resin were determined by GC using the following method. The resin sample was absorbed onto an Extrelut column (available from EM Science, Extrelut QE, Part #901003-1) and ethyl acetate was passed through the column for extraction. A portion of the ethyl acetate solution was chromatographed on a wide-bore capillary column. If using a flame ionization detector (FID), these components were quantified using n-octanol as an internal standard. If using an electrolyte conductivity detector (ELCD) or a halogen-specific detector (XSD), use an external standard method using peak-matching quantification. The data system is either Millennium2010 or HP ChemStation. The FID detector was purchased from Hewlett-Packard (HP) as part of the 5890 GC. ELCD detector, model 5220, was purchased from OI Analytical. The XSD detector was purchased from OI Analytical, model 5360 XSD. The GC instrument used was an HP Model 5890 Series II. The column was DB-WAX (Megabore, J&W Scientific, Inc.) 30 m x 0.53 mm with a film thickness of 1.5 microns. For the FID and ELCD, the carrier gas was helium at a flow rate of 10 mL/min. The oven program was 35°C for 7 minutes followed by 8°C/minute ramp to 200°C and hold at 200°C for 5 minutes. The FID uses 30 mL/min of hydrogen and 250°C, 400 mL/min of air. The ELCD uses n-propanol as the electrolyte, and the electrolyte flow rate is set at 50%, and the reactor temperature is 900 °C. The XSD reactor was operated at 1100°C in the oxidized state with a high purity air flow rate of 25 mL/min.

Furthermore, paper products containing the resins of the present invention can be stored without improperly formed CPD. Thus, the paper products of the present invention can have initially low levels of CPD and can maintain low levels of CPD over extended storage periods. More specifically, paper products of the present invention, made with an additional amount of 1% by weight resin, will contain less than about 250 parts per billion ( ppb), more preferably less than about 100 ppb of CPD, even more preferably less than about 50 ppb of CPD, even more preferably less than about 10 ppb of CPD, even more preferably less than about 1 ppb of CPD. Furthermore, paper products of the present invention, made with an additional amount of 1 wt% resin, exhibit a CPD content increase of less than about 250 ppb, more preferably less than about 100 ppb when stored for 2 weeks, preferably at least 6 months, even more preferably at least 1 year , even more preferably less than about 50 ppb CPD, even more preferably less than about 10 ppb CPD, even more preferably less than about 1 ppb CPD. In other words, the paper product of the present invention has storage stability and will not generate excessive CPD content in the paper product when the paper product is stored as short as 1 day to as long as 1 year. Accordingly, the resins of the present invention form minimal CPD in paper products, especially those exposed to aqueous environments, especially hot aqueous environments, such as tea bags, coffee filters, and the like. Other examples of paper products include paperboard grades for packaging and facial tissue and decal paper grades.

Paper can be made by adding additional levels of resin other than about 1% by weight; however, the CPD content should be calibrated for this additional level. For example, for a paper product having a measured CPD content of 50 ppb by addition at 0.5 wt % add-on, the calibrated CPD based on 1 wt % add-on would be 100 ppb (50 ppb/0.5 % add-on).

For the determination of CPD in paper products, the paper products were extracted with water according to the method described in European Standard EN 647, dated October 1993. Then 5.80 g of sodium chloride was dissolved in 20 ml of the aqueous extract. The salinized aqueous extract was transferred to a 20 g capacity Extrelut column and the column was saturated for 15 minutes. After washing 3 times with 3 ml ethyl acetate and saturating the column, the Extrelut column was eluted until 300 ml of eluate was recovered in about 1 hour. Concentrate 300 ml of the ethyl acetate extract to about 5 ml using a 500-ml Kuderna-Danish concentrator (use a miniature Kuderna-Danish device for further concentration if necessary). The concentrated extract was analyzed by GC using the procedure and apparatus described above. Typically, an electrolyte conductivity detector (ELCD) or a halogen specific detector (XSD) is used. Other sensitive detectors can be used, such as electron capture detectors. Alternatively, the procedure described in Example 4 can be used to determine CPD in paper products.

Resins that may be treated with the enzymatic reagents of the present invention may include any polyamine-epihalohydrin resin. The present invention also relates to polyamine-epihalides prepared by reacting an epihalohydrin, such as epichlorohydrin, with a prepolymer (also referred to herein interchangeably as a polymer), such as a polyaminoamide prepolymer. Preparation, use and disposal of alcohols such as polyaminopolyamide-epichlorohydrin. In the case of polyaminopolyamide resins, it should be noted that polyaminoamide prepolymers are also known as polyamidoamines, polyaminopolyamides, polyamidopolyamines, polyamidepolyamines, polyamides, alkaline Polyamide, cationic polyamide, aminopolyamide, amidopolyamine or polyaminoamide.

Preferred polymers for use in the present invention include cationic polymers, alone or with other polymers. Particularly preferred cationic polymers include those used to impart wet strength to paper and creping agents. A list of many polymers useful in papermaking formulations, such as wet strength and creping agents, is described in Paper Chemistry, ISBN 0-216-92909-1 pp. 78-96, published in the USA by Chapman Hall, New York. Chapter 6 of that book is entitled "Wet Strength Chemistry" and is incorporated herein by reference in its entirety. Chapter 6 describes several classes of polymers used to impart wet strength to paper, including: polyaminoamide-epichlorohydrin resins, urea-formaldehyde resins, melamine-formaldehyde resins, epoxidized polyamide resins, acetaldehyde Acidified polyacrylamide resins, polyethyleneimine resins, dialdehyde starches, protein binders treated with formaldehyde, cellulose xanthate (viscose), synthetic latex, vegetable gums, and glyoxal. The polyaminoamide-epichlorohydrin can be Kymene® brand polyaminoamide-epichlorohydrin resin, such as Kymene® 557LX, Kymene® SLX2 or Kymene® 617, or a polyamine-epichlorohydrin such as Kymene® 2064, Kymene® 367 resin and Kymene 736 or a polyamide-polyureidene-epihalohydrin resin such as Kymene 450.

The present invention relates to cationic polymers, such as polyamine-epichlorohydrin resins, which can be used alone or in combination with other polymers as wet strength and creping agents for paper. These resins include epichlorohydrin resins and nitrogen-containing cationic polymers, both derived from epichlorohydrin reactants. Preferred resins for the purposes of the present invention include polyaminoamide-epichlorohydrin wet strength resins such as US2926154, 3332901, 3891589, 3197427, 4240935, 4857586; , 09/363224 and 09/330200. Furthermore, resins include Crepetrol® 80E or Crepetrol® A3025, Crepetrol® A6115, Crepetrol® A8225, Crepetrol® 870, SPC 003 and Rezosol® 8289 creping agents, all available from Hercules Incorporated, Wilmington, Delaware. It is noted that these resins are generally referred to herein as polyamine-epihalohydrin resins, and that these resins include, but are not limited to, polyaminopolyamide-epihalohydrin resins (also known as polyaminoamide-epihalohydrin resins) Halohydrin resins, polyamide polyamine-epihalohydrin resins, polyamine polyamide-epihalohydrin resins, aminopolyamide-epihalohydrin resins, polyamide-epihalohydrin resins); polyalkylenes based polyamine-epihalohydrin; and polyaminoureide-epihalohydrin resins, copolyamide-polyureide-epihalohydrin resins, polyamide-polyureide-epihalohydrin resins, The preferred epihalohydrin in each instance is epichlorohydrin. Methods for the preparation of these known resins are also disclosed in these documents, which are incorporated herein by reference in their entirety.

The epichlorohydrin resins exemplified in these patents are characterized by the presence of N-chloropropane groups of the formula:

and a quaternary N-chloropropane group of the formula:

where the tetrasubstituted nitrogen atom is positively charged (quaternary nitrogen) and is therefore a cation;

and isomeric 3-hydroxyazetidinium chloride groups of the formula:

Preferred cationic polymers for use in the present invention are polymers having the formula:

The tetrasubstituted nitrogen atoms marked with an asterisk therein are positively charged (quaternary nitrogen) and are therefore cationic. The nitrogen atom is in a 4-membered ring (ie, 3-hydroxyazetidinium group). Other uncharged polymer units are also co-present in the polymer chains of such resins. Even small amounts of negatively charged (ie, anionic) groups can be present on the polymer, but the net charge along the polymer chain is positive. X- is a simple anion which is not covalently attached to the polymer chain. Usually the anion is chloride ion, and n is an integer of about 5-several thousand, preferably 5-3000.

Creping agents include, but are not limited to, Crepetrol(R) 80E or Crepetrol(R) A3025, Crepetrol(R) A6115, Crepetrol(R) A8225, Crepetrol(R) 870, SPC 003 and Rezosol(R) 8289 creping agents.

For wet strength agents, preferred resins include those formed in polyamide-epihalohydrin reactions having a molar ratio of epihalohydrin to secondary amino groups of less than 1, more preferably epihalohydrin, although ratios greater than 1 may be used. The molar ratio of alcohol to secondary amino groups is less than about 0.975, wherein the preferred range of molar ratio of epihalohydrin to secondary amino groups is about 0.5-0.975, more preferably the molar ratio of epihalohydrin to secondary amino groups is about 0.6 -0.975, even more preferably about 0.8-0.975. For creping agents, preferred resins include polyamide-epihalides in a molar ratio of epihalohydrin to secondary amino groups of less than about 0.50, more preferably less than about 0.25, and may even be less than 0.1, preferably with a lower limit of about 0.05 Resins formed in alcohol substitution reactions.

Furthermore, the creping agents of the present invention do not require as many crosslinking functional groups as in wet strength agents, and therefore can have lower azetidinium contents than wet strength agents. Accordingly, preferred creping agents have an azetidinium content of less than about 10 mole percent, with a preferred range of about 5-10 mole percent, and preferred wet strength agents have an azetidinium content of greater than about 30 mole percent, with a preferred range of about 30-70 mole percent %. The mol% of azetidinium and the mol% of other substances can be determined by the following NMR procedure.

NMR steps

Its ¹³ CNMR spectrum was obtained using a BRUKER AMX spectrometer equipped with a 10 mm broadband probe. A ¹³ C NMR operating frequency of 100 MHz (AMX400) or 125 MHz (AMX500) was sufficient for data collection. In both cases, spectra with continuous ¹ H decoupling were obtained. Electronic integration of the appropriate signals provides the molar concentrations of the following alkylation components: ACH, EPX, GLY and AZE.

Among them: ACH = polymeric aminochloropropanes, EPX = polymeric epoxides, GLY = polymeric glycols, AZE = azetidinium ions

In order to calculate the respective concentrations of these species, integer values must be placed on a 1 carbon basis. For example, the spectral region of 20-42 ppm represents the 6 carbons in the diethylenetriamine-adipate backbone, so the integer value is divided by 6. This value was used as the polymer common denominator (PCD) for the calculation of alkylated species. The chemical shifts of these species are provided below (using an acetonitrile field reference of 1.3 ppm). Use the corresponding integer value for each alkylation product for numerator calculations, see examples below:

- ACH signal at 68-69ppm represents 1 carbon;

Integer of ACH÷PCD=Mole parts ACH

- GLY signal at 69-70ppm represents 1 carbon;

Integer of GLY÷PCD=Mole parts GLY

- EPX carbon at 51-52ppm represents 1 carbon;

Integer of EPX÷PCD=Mole parts EPX

- AZE signal at 73-74ppm represents 2 carbons, therefore, a split factor of 2 is required;

Integer of AZE/2÷PCD = molar parts AZE

The following spectroscopic parameters are standard test conditions ^for13C NMR analysis of Kymene resins or creping agents on a Bruker AMX400. temperature 25°C Resonance frequency 100MHz #data point 64K dwell time 20 microseconds collection time 13 seconds scan width 25000Hz scan numbers 1K relaxation delay 3 seconds Pulse tip cone angle 70° pulse program zgdc processed spectral size 64K apodization function index line broadening 3Hz

Also in accordance with the present invention, for creping agents derived from prepolymers containing tertiary amine functionality, the creping agent will preferably have a content of less than about 30 mole percent quaternary aminohalopropanes, such as aminochloropropanes, whereas the present invention The inventive wet strength agent preferably has a quaternary aminohalopropane such as aminochloropropane in a content greater than 30 mol %. Also, without wishing to be bound by theory, it is believed that secondary amine compounds, such as diethylenetriamine, form azetidinium groups, while tertiary amine compounds, such as methylbis(3-aminopropyl)amine, Formation of quaternary aminochloropropane groups. Examples of tertiary amine compounds include, but are not limited to, the reaction product of adipic acid and methylbis(3-aminopropyl)amine, resulting in a tertiary amine prepolymer. This prepolymer is used to prepare tertiary amine-based resins containing quaternary aminohalopropane groups.

The preferred polyamines of the present invention are obtained by dicarboxylic acid or its derivatives with methyl bis(3-aminopropyl)amine or with 2-4 alkylene groups with 2-4 carbons, 2 primary It is produced by reacting polyalkylene polyamines with amine groups and 1-3 tertiary amine groups. Dicarboxylic acid derivatives suitable for use in preparing the polyaminoamides include esters, anhydrides and acid halides.

Procedures for the preparation of polyaminoamides from polyalkylene polyamines are described in US 2,926,154 to Keim (herein incorporated by reference in its entirety). Procedures for the preparation of polyaminoamides using methylbis(3-aminopropyl)amine are described in US5338807 and US5994449 to Espy et al. (incorporated by reference in their entirety).

By expansion of the above, polyaminopolyamide-epichlorohydrin resins include epichlorohydrin and polyamides derived from polyalkylene polyamines and saturated aliphatic dicarboxylic acids containing from about 2 to about 10 carbon atoms water-soluble polymer reaction product. Such resins have been found to impart wet strength to paper whether produced under acidic, basic or neutral conditions. Also, these resins are essentially cellulosic fibers so that they can be applied economically, while the fibers are dilute aqueous suspensions for paper mill consistency.

In preparing the cationic resins intended for use herein, dicarboxylic acids are first reacted with polyalkylenepolyamines under conditions such as to produce water-soluble polyamides containing cyclic groups as follows:

-NH(C _n H _2n NH) _x -CORCO-

wherein n and x are each 2 or more, and R is a divalent hydrocarbon group of a dicarboxylic acid. This water-soluble polyamide is then reacted with epihalohydrin to form a water-soluble cationic resin. The dicarboxylic acids intended to be used in the preparation of the resins of the present invention are saturated aliphatic dicarboxylic acids containing 2 to 10 carbon atoms such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, azelaic acid, Diacid etc. Saturated dibasic acids having 4 to 8 carbon atoms in the molecule, such as adipic acid and glutaric acid, are preferred. It is also possible to use mixtures of two or more of said saturated dicarboxylic acids. Derivatives of dicarboxylic acids, such as esters, half-esters and anhydrides, can also be used in the present invention, such as dimethyl adipate, diethyl adipate, dimethyl glutarate, diethyl glutarate, ethyl ester, dimethyl succinate and diethyl succinate. Mixtures of two or more derivatives of dicarboxylic acids and mixtures of one or more derivatives of dicarboxylic acids with dicarboxylic acids may also be used.

Various polyalkylene polyamines can be used, including polyethylene polyamines, polypropylene polyamines, polybutylene polyamines, polypentylene polyamines, polyhexylene polyamines, etc. and mixtures thereof, wherein said polyethylenepolyamines represent an economically preferred class. More specifically, the polyalkylenepolyamines intended to be used can be represented by polyamines in which the nitrogen atoms are chained together by groups of the formula _-CnH2n- , where n is _a unit greater than these groups in the molecular weight and small integers of quantities, from 2 to about 8. The nitrogen atoms may be bonded to adjacent or distant carbon atoms in _the group _-CnH2n- , but not to the same carbon atom. The present invention includes not only the use of the following polyamines, which are available in suitably pure form: diethylenetriamine, triethylenetetramine, tetraethylenepentamine and dipropylenetriamine, but also mixtures and various crude polyamine materials. For example, a mixture of polyethylene polyamines obtained by reacting ammonia with ethylene dichloride, refined only to the extent that chlorides, water, excess ammonia and ethylenediamine are removed, is a satisfactory starting material. The term "polyalkylenepolyamine" as used in the claims thus refers to and includes any of the polyalkylenepolyamines referred to above or mixtures of these polyalkylenepolyamines and their derivatives. Other polyamines suitable for use in the present invention include: bis-hexamethylenetriamine (BHMT), methyldiaminopropylamine (MBAPA), other polyalkylene polyamines (eg spermine, spermidine). Preferred polyamines are diethylenetriamine, triethylenetetramine, tetraethylenepentamine and dipropylenetriamine.

In some cases it will be desirable to increase the spacing of the secondary amino groups on the polyamide molecule in order to alter the reactivity of the polyamide-epichlorohydrin complex. This can be achieved by substituting a portion of the polyalkylenepolyamine with a diamine such as ethylenediamine, propylenediamine, hexamethylenediamine, and the like. For this purpose, up to about 80% of the polyalkylenepolyamines can be replaced by equimolar amounts of diamines. Typically, about 50% or less will be replaced for this purpose.

Suitable aminocarboxylic acids or their lactams containing at least 3 carbon atoms are also suitable for increasing the spacer according to the invention. For example, 6-aminocaproic acid and caprolactam.

Polyurethane-epihalohydrin resins, especially polyureidene-epichlorohydrin resins, are also included in the present invention, such as disclosed in US4487884 and 3311594 (which are incorporated herein by reference in their entirety), For example, Kymene(R) type 450 resin (available from Hercules Incorporated, Wilmington, Delaware). The polysemicarbazide-based resins intended for preparation and for use herein are prepared by reacting epichlorohydrin with polysemicarbazide groups containing free amino groups. These polyurethanes are water-soluble substances containing tertiary amino groups and/or mixtures of tertiary amino groups with primary and/or secondary amino groups and/or quaternary ammonium groups. However, tertiary amino groups should account for at least 70% of the basic nitrogen groups present in the polysemicarbazide groups. These polysemicarbazide groups can be prepared by reacting urea or thiourea with polyamines containing at least 3 amino groups, at least one of which is a tertiary amino group. The reaction can be carried out in a suitable solvent such as xylene, if desired.

The polyamine reactant should preferably have at least 3 amino groups, at least one of which is a tertiary amino group. The polyamine reactant may also have a limited amount of secondary amino groups. Typical polyamines of this type suitable for use as described herein above are methylbis(3-aminopropyl)amine (MBAPA), methylbis(2-aminoethyl)amine, N-(2-aminoethyl) ) piperazine, 4,7-dimethyltriethylenetetramine, etc., which are available in reasonably pure form, but also as mixtures of various crude polyamine materials.

To prepare the prepolymer from the diacid and the polyalkylene polyamine, the reaction mixture is heated at atmospheric pressure, preferably at a temperature of about 125-200° C., preferably for about 0.5-4 hours. When reduced pressure is used, low temperatures such as 75°C to 150°C may be used. This polycondensation reaction produces water as a by-product, which is removed by distillation. At the end of the reaction, the resulting product was dissolved in water at a concentration of about 50% by weight of total polymer solids.

When diesters are used instead of diacids, the prepolymerization can be carried out at atmospheric pressure and at lower temperatures, preferably about 100-175°C. The by-product in this case will be an alcohol, the type of alcohol depending on the identity of the diester. For example, when dimethyl esters are used, the alcohol by-product is methanol, while ethanol is a by-product obtained from diethyl esters. When reduced pressure is used, lower temperatures such as 75°C to 150°C may be used.

In converting the polyamide formed as described above into a cationic resin, it is mixed with epoxy at a temperature of from greater than about 0°C, more preferably about 25°C, to about 100°C, and preferably about 35°C to about 70°C. The chloropropane is reacted until the viscosity of a 20% solids solution at 25°C reaches about C or higher on the Gardner Holdt scale. In order to moderate the reaction, it is preferable to carry out the reaction under an aqueous solution. Although not required, pH adjustments can be made to increase or decrease the rate of crosslinking.

When the desired viscosity is reached, enough water can be added to adjust the solids content of the resin solution to the desired amount, i.e. around 15 wt%, the product can be cooled to about 25°C, and then its pH can be adjusted by adding enough acid Reducing to below about 6, preferably below about 5, most preferably below about 4 increases gel stability for stable storage. The product may be stabilized with any suitable inorganic or organic acid, such as hydrochloric, sulfuric, methanesulfonic, nitric, formic, phosphoric, and acetic acids. Preference is given to halogen-free acids, such as sulfuric acid.

The composition of the present invention is used to crepe a fibrous web by: (1) applying the composition described above to the dry surface of the web or to the web; (2) pressing the fibrous web against its dry surface to effectively bonding the web to the dry surface; and (3) creping the fibrous web by removing the web from the dry surface with a creping device, such as a doctor blade. Preferably in step (1), the composition is applied to the dry surface of the web. A preferred fibrous web is a cellulose web.

Preferably, the creping adhesive is applied as an aqueous solution containing from about 0.1 to about 10% by weight of the resin composition. More preferably the resin composition is a solution in an amount of about 0.25 to about 5 wt%, most preferably about 0.5 to about 2 wt%. For creping agents on a dry weight basis, a minimum amount of about 0.001% by weight based on the dry weight of the pulp or paper is used. A more preferred minimum amount is about 0.005 wt%, and a most preferred minimum amount is about 0.01 wt%. The preferred maximum amount of resin composition is about 2 wt%. A more preferred maximum amount is about 1 wt%, and a most preferred maximum amount is about 0.5 wt%. The dry surface most commonly used in industrial operations is the Yankee dryer, and the aqueous solution of the binder is most often applied by spray to the creping drum or drum. Alternatively, however, it may be added by application onto the fibrous web, preferably by spraying. In the case of a cellulosic web, ie paper, the creping binder can be added by application to the wetted web at the end of wetting on the paper machine. In some cases a creping adhesive may be added to the pulp prior to forming the sheet.

Other components, especially agents that improve the adhesion of the web to dry surfaces, may be used in conjunction with the creping adhesives of the present invention.

These agents, also known as release agents or plasticizers, include water-soluble polyols, glycols, polyethylene glycols, sugars, oligosaccharides and hydrocarbon oils.

The method for making paper using the resin composition of the present invention comprises: (a) providing an aqueous pulp suspension; (b) adding resin to the aqueous pulp suspension, and (c) liquidating the aqueous pulp suspension produced in (b) sheets and dried to obtain paper.

The aqueous pulp suspension of step (a) of the process is obtained by means known in the art, such as known mechanical, chemical and semi-chemical pulping methods. Typically, after the mechanical pulverization and/or chemical beating steps, the pulp is washed to remove residual beating chemicals and to dissolve wood components. Bleached or unbleached pulp fibers can be used in the process of the invention. Recycled pulp fibers are also suitable.

In step (b) the resin of the present invention is preferably added to the pulp slurry in a minimum amount of about 0.1% by weight based on the dry weight of the pulp. A more preferred minimum amount is about 0.2 wt%. A preferred maximum amount of the resin composition is about 5 wt%, a more preferred maximum amount is about 3 wt%, and a most preferred maximum amount is about 1.5 wt%. The resin composition is usually added in a water-soluble form. In addition to resins, other substances commonly used in paper can also be added. These include, for example, sizing agents, pigments, alum, brighteners, dyes and dry strength agents, added in amounts known in the art.

Step (c) is carried out according to procedures known to those skilled in the art of papermaking.

As discussed above, a resin having at least reduced CPD formation may be a resin produced in a resin synthesis process without further processing. Also, the resin can be treated by various methods prior to reducing and/or removing CPD forming species. Also, after reducing and/or removing CPD forming species, the resin can be treated by various methods. Furthermore, the resin can be treated differently prior to the reduction and/or removal of the CPD forming species, and the resin can also be treated differently after the reduction and/or removal of the CPD forming species. For example, the resin can be treated by various methods such as methods to remove low molecular weight epihalohydrins and epihalohydrin by-products such as epichlorohydrin and epichlorohydrin by-products such as CPD in the resin solution. Without limiting the treatment or available resins, note that base ion exchange columns such as disclosed in US5516885 and WO 92/22601; carbon adsorption as disclosed in WO 93/21384; such as U.S. Statutory Invention Registration H1613 Membrane separations such as ultrafiltration, extraction such as ethyl acetate; or biological dehalogenation to reduce or remove CPD forming species such as disclosed in US5972691, WO 96/40967 and US5470742, 5843763 and 5871616 and US application 09/629629 Pre and/or post treatment resins such as Kymene SLX2, Kymene 617 and Kymene 557LX (available from Hercules Incorporated, Wilmington, Delaware), and Crepetrol 80E or Crepetrol A3025, Crepetrol A6115, Crepetrol A8225, Crepetrol 870, SPC 003 and Rezosol 8289 creping agents. The contents of the respective disclosures of these documents are incorporated herein by reference in their entirety. Furthermore, any combination of the reduction or removal of CPD-forming species disclosed in the above-cited US Patent Applications 09/592681, 09/363224, and 09/330200 may be used with enzymatic treatments that reduce and/or remove CPD-forming species.

Furthermore, according to the present invention, it is also noted that the enzymatic treatment to remove or reduce CPD forming substances can be carried out in an overlapping manner with biological dehalogenation, or can be carried out simultaneously with biological dehalogenation. Therefore, the present invention also relates to a combined method in which enzymatic release of the resin 3-CPD is initiated and the reduction of nitrogen-free organohalogen compounds is carried out simultaneously.

It should also be noted that, in addition to the enzymatic treatment following the biological dehalogenation treatment, it is also possible to carry out both treatments simultaneously (known as combined treatment). "Simultaneously" means that the second treatment (or biological dehalogenation or enzymatic treatment) can start before the first treatment (or biological dehalogenation or enzymatic treatment) is completed. For the purposes of the present invention, the desired viscosity is achieved by balancing the conditions: time, temperature, pH, enzyme concentration, onset viscosity and solids content. For example, the combination treatment can start at pH 6.8-7.8 for the first 4-24 hours and decrease to pH 5.5-7.0, or the pH can drop to 6.5-7.2 within the last 8-48 hours of the combination treatment. Preferred combined treatment conditions include, but are not limited to, pH 6.5-8.0, more preferably pH 6.8-7.6; preferred temperature range is 20°C-35°C, more preferably 25°C-33°C. The enzyme concentration for the combined treatment conditions will depend on its activity. For example, in the case of ALCALASE, the enzyme may be present in the following amount: about 1 g of ALCALASE 2.5L Type DX (used as is)/1600 g polyamine-epichlorohydrin resin dry basis - 1 g of ALCALASE 2.5L Type DX (used as is) )/1.5g polyamine-epichlorohydrin resin dry basis, the enzyme may also be present in the following amount: about 1g of ALCALASE 2.5L type DX (used as is)/160g polyamine-epichlorohydrin resin dry basis-1g of ALCALASE 2.5L Type DX (used as is)/4g polyamine-epichlorohydrin resin dry basis. Preferably the combination treatment is completed in 48 hours or less. Most preferably the combination treatment is completed in 24 hours or less. For creping aids, the solids content may be less than about 15 wt%, typically 4 to 14.5 wt%, preferably about 8 wt% to about 14.5 wt%, when using the combination of processing conditions. Combination treatments of creping aids may also be carried out at solids levels of 15 wt% and above, with a preferred total solids content of 15-40 wt%, preferably 18-35 wt%, even more preferably 18-28 wt%. Another range that can be used in the present invention is 15-30 wt%.

The preferred total solids content is 15-40 wt%, preferably 16-35 wt%, even more preferably 18-28 wt%, when combined processing conditions are used for wet strength resins of 15 wt% or greater. Preferred ranges when combining less than 15 wt% wet strength resin are from about 4 wt% to about 14.5 wt%, and from about 8 wt% to about 14.5 wt%.

Furthermore, the present invention enables biodehalogenation to be performed at high total solids content, and combined enzymatic treatment to remove or reduce CPD forming species, and to perform biodehalogenation at high total solids content, which reduces process cycle time while Optimal use of reactor volume results when the process is carried out in batch or (repeated) feed mode.

Biodehalogenation can be carried out in various ways, with or without prior inorganic alkali treatment, such as disclosed in any of US5470742, 5843763 and 5871616 and US application 09/629629, or as disclosed in US5972691 and WO 96/40967 Pre-alkaline treatment and biological dehalogenation, wherein the resin composition can be reacted with an appropriate amount of microorganisms or enzymes to treat the epihalohydrin hydrolyzate to a low level. Microorganisms use dehalogenases to release halide ions from epihalohydrins and halohydrins, and then use other enzymes to finally decompose the reaction products into carbon dioxide and water.

While not wishing to be bound by theory, it is noted that when CPD forming species are removed or reduced, CPD is released from the oligomeric and/or polymeric components of the resin, and thus CPD is a component of the resin solution. In this concept, the resin is preferably treated to remove or reduce CPD forming species, and then the resin is biodehalogenated. In this way, epihalohydrins and epihalohydrin hydrolysates (also referred to as hydrolysis by-products), including released CPD, can be removed, for example by biological dehalogenation. However, the resin may be initially treated, for example by biodehalogenation, and then treated to remove, inhibit and/or reduce CPD forming species. In particular, any CPD released by processing should be readily soluble and thus be at least partially washable from the resin. For example, when a resin with released CPD is contained in a paper product, the CPD can be at least partially washed out of the paper product, and as a result of this treatment, the resin in the paper product will not generate CPD or will not generate unwanted amounts of CPD. Also, as discussed above, enzymatic treatment to remove or reduce CPD forming species can be performed in an overlapping/simultaneous manner with biological dehalogenation.

Biocatalysts can be provided as living cells or as immobilized, unrefined cell-free extracts or refined dehalogenases. The term "biodehalogenation" refers to the dehalogenation of nitrogen-free organohalogen compounds using a biological catalyst.

As a biocatalyst capable of biological dehalogenation, any microorganism capable of dehalogenating nitrogen-free biological dehalogenation compounds, preferably CPD and DCP, can be used, while the nitrogen-containing cationic polymer Basically remain unaffected. Preferably, the microorganisms used are Agrobacterium radioactive (HK7) or Arthrobacter histidinophila (HK1 ), and preferably a two-component mixture of Agrobacterium radioactive (HK7) or Arthrobacter histidinophila (HK1) is used. Preference is given to using a single microorganism HK1 when only CPD is present. When only DCP is present, it is preferred to use a single microorganism HK7. Although the enzymes that make this process operable have not been precisely identified, it is believed that the enzymes that perform this process belong to a class of enzymes known as "hydrogen halide lyase-like dehalogenases".

In particular, a number of strains are disclosed in US5470742, 5843763, 5871616 and 5972691 and US application 09/629629, the contents of which are incorporated herein by reference. These strains include microorganisms containing dehalogenases capable of dehalogenating halohydrins and epihalohydrins, deposited under NCIMB deposit accession numbers 40271, 40272, 40273, 40274, 40313 and 40383. NCIMB stands for "National Collection of Industrial and Marine Bacteria". NCIMB, located at 23 St. Machar Drive, Aberdeen AB21RY, Scotland, UK, is an organization in the UK responsible for certifying and retaining bacterial samples submitted for patent applications. In patent matters, NCIMB will provide authentic samples of bacteria claimed in patent documents to interested parties upon request. NCIMB 40271 (Arthrobacter spp.), 40272 (Agrobacterium radioactive HK7), 40273 (Burkholderia cepacia, formerly known as Pseudomonas cepacia) and 40274 (Arthrobacter histidinophilus HK1) are deposited at April 2, 1990. NCIMB 40383 (Rhodococcus sp.) was deposited on March 11, 1991, and NCIMB 40313 (Burkholderia cepacia, formerly known as Pseudomonas cepacia) was deposited on August 30, 1990. Accordingly, these microorganisms have been deposited in the depository under the Budapest Treaty, and these strains will be made available to the public irrevocably and without restriction or condition when the patent is published.

Moreover, it is noted that two bacterial strains are preferably used, which were isolated from soil samples and jointly named HKC, Arthrobacter histidinophila (HK1) (deposited at the Centraalbureau voor Schimmelcultures at Oosterstraat 1 on July 10, 2000, P.O. Box 273, 3740 AG BAARN, The Netherlands, with accession number CBS 108919) and Agrobacterium radioactive (HK7) (deposited at Centraalbureau voorSchimmelcultures at Oosterstraat 1, P.O. Box 273, 3740 AG BAARN, The Netherlands, on 10 July 2000, registered No. CBS 108920). In patent matters, the Centraalbureau voor Schimmelcultures, a depository complying with the Budapest Treaty, will provide interested parties upon request with samples of bacteria claimed in patent documents. Accordingly, these microorganisms have been deposited in the depository under the Budapest Treaty, and these strains will be made available to the public irrevocably and without restriction or condition when the patent is published. Note that NCIMB 40272 and CBS 108920 are believed to be the same microorganism, and NCIMB 40274 and CBS 108919 are believed to be the same microorganism.

Each of these microorganisms is capable of degrading 1,3-DCP and 3-CPD. Furthermore, radioactive Agrobacterium (HK7) was able to reduce 1,3-DCP levels faster than Arthrobacter histidophagae HK1, which showed preferential degradation of 3-CPD. Therefore, as disclosed in US application 09/629629, it is assumed that the best biodehalogenation performance is obtained when both bacteria are present. To ensure that both bacteria are present in the biodehalogenation process, it is preferred to start the process with radioactive Agrobacterium (HK7), followed by the addition of Arthrobacter histidinophila (HK1). This is especially the case for starting a continuous biodehalogenation process.

Microorganisms containing suitable enzymes are used for the dehalogenation of epihalohydrin hydrolysates contained in the resin composition with or without an initial inorganic base treatment. Enzymes and microorganisms are maintained at suitable concentrations to metabolize the hydrolyzate substantially to chloride ions and ultimately to carbon dioxide and water. Therefore, the hydrolyzate concentration in the resin composition of the present invention is preferably lower than about 100 ppm (parts per million by weight of the total weight of the resin-containing aqueous solution after the relative biological reaction step) after biological dehalogenation treatment, more preferably lower than about 50 ppm ( Parts per million by weight of the total weight of the resin-containing aqueous solution after the relative biological reaction step), more preferably less than about 10 ppm (parts per million by weight of the total weight of the resin-containing aqueous solution after the relative biological reaction step), more preferably low At about 5 ppm (parts per million by weight of the total weight of the resin-containing aqueous solution after the relative biological reaction step), even more preferably less than about 1 ppm (parts per million by weight of the total weight of the resin-containing aqueous solution after the relative biological reaction step) ).

To this end, the concentration of microorganisms should be at least about 5 x ¹⁰⁷ cells/ml, preferably at least about ¹⁰⁸ cells/ml, most preferably at least about ¹⁰⁹ cells/ml. To maintain an optimal active content of cells in the reactor, the reaction is best performed in a stirred tank reactor at about 30°C +/- 5°C in the presence of oxygen (eg about 5 to about 100% DOT) and nutrients. As used herein, the term "DOT" means "dissolved oxygen tension" and is the amount of oxygen dissolved in a given volume of water relative to oxygen-saturated water at the same temperature and pressure, expressed as a percentage. In a continuous process, residence time is controlled by flow rate and monitored to ensure complete reaction. Thus, the concentration of epihalohydrin hydrolyzate in the reactor at steady state will be from about 1 to about 1000 ppm. In batch or feed mode, they may preferably be repeated, with monitoring, for example by GC analysis, ensuring complete reaction to obtain the desired reduced amount of epihalohydrin hydrolyzate.

The biological dehalogenation process of the present invention is carried out by contacting a microorganism or a cell-free enzyme-containing extract with an aqueous composition containing unwanted organohalogen contaminants. Such contacting is typically accomplished by forming a slurry or suspension of the microorganism or cell-free extract in the aqueous composition with vigorous agitation.

Microorganisms or enzymes may, if desired, be removed from the product stream by filtration, sedimentation, centrifugation or other means known to those skilled in the art. Alternatively the microorganisms or enzymes may remain in the final product and optionally be inactivated by heat sterilization (for example by treatment at 140°C for 20 seconds) or by addition of a suitable biocide at a suitable concentration. Suitable biocides can be readily selected by one of ordinary skill in the art. Thus, microorganisms can be inactivated by lowering the pH of the aqueous mixture to 2.8 followed by the addition of a sufficient amount (typically 0.02% to 0.1% by weight of the aqueous composition) of a proprietary biocide (e.g., Proxell® BD biocides, including 1,2-benzisothiazolin-3-one). The biocide can be added with potassium sorbate.

Removal of microorganisms may be performed by one or more steps of filtration, centrifugation, sedimentation or any other known technique for removing microorganisms from a mixture. The microorganisms mineralize nitrogen-free organohalogen compounds, producing CO ₂ , water and biomass, leaving no glycerol in the resin. When the biocatalyst is an immobilized dehalogenase, the reaction product is glycidol, which can be converted to glycerol with an immobilized lyase.

The problem associated with the removal of microorganisms from the mixture is that strength separation methods, such as microfiltration, remove not only the microorganisms but also the cationic polymer particles, resulting in reduced wet strength properties, which is undesirable. Therefore, it is preferred to leave the inactivated microorganisms in the mixture to avoid the problem of reduced wet strength properties.

Surprisingly it has been determined that when the resin contains a tertiary amine-based resin, such as Kymene® 450, Crepetrol® A3025 or Crepetrol® 80E, there is a combination of resins with a high concentration of solids, i.e. greater than 15 wt%, more preferably greater than 20 wt%, preferably greater than 25 wt% Substances can undergo biological dehalogenation using microorganisms and/or enzymes. In the past, secondary amine-based resins such as Kymene(R) 557H, Kymene(R) 557LX, Kymene(R) SLX, Kymene(R) Plus were not effective in biodehalogenation at solids concentrations of 15 wt% or greater. In the present invention, the secondary amino-based resin can be effectively biodehalogenated at 15 wt% or more. In addition, it has been found that Daniels resins can biodehalogenate at 15 wt% or greater.

In the case of Daniel's resins, it should be noted that resins derived from epihalohydrins such as epichlorohydrin and polyalkylenepolyamines such as ethylenediamine (EDA), bis-hexamethylenetriamine (BHMT) and Reactive cationic water-soluble resins of hexamethylenediamine (HMDA) have been known for a long time. These polyalkylene polyamine-epihalohydrin resins are described, for example, in J.M. Baggett et al. US Patent No. 3,655,506, and others such as Daniel et al. The disclosures of these patents are incorporated herein by reference.

The polyalkylene polyamines used in the present invention can preferably be selected from polyalkylene polyamines of the following formula:

H ₂ N-[CH ₂ -CHZ-(CH ₂ ) _n -NR-] _x -H

Wherein: n=1-7, x=1-6, R=H or CH ₂ Y, Z=H or CH ₃ , and Y=CH ₂ Z, H, NH ₂ or CH ₃

Polyalkylene polyamines of the formula:

H ₂ N-[(CHZ) _m -(CH ₂ ) _n -NR-] _x -H

Where: m=1-6, n=1-6, and m+n=2-7, R=H or CH ₂ Y, Z=H or CH ₃ , and Y=CH ₂ Z, H, NH ₂ or CH ₃ , and mixtures thereof.

The polyalkylenepolyamine-epihalohydrin resins contain the water-soluble polymerization reaction product of epihalohydrin and polyalkylenepolyamine. The polyalkylene polyamine is added to the aqueous mixture of epihalohydrin in the preparation of Daniel's resin so that the temperature of the mixture does not exceed 60°C during the addition. Lower temperatures allow for further increases, although too low temperatures dangerously build up potential reactivity in the system. A preferred temperature is from about 25°C to about 60°C. More preferably from about 30°C to about 45°C.

Depending on the relative amounts of epihalohydrin and polyamine, alkylation of the polyamine will proceed rapidly to form secondary and tertiary amines. The amount of epihalohydrin and polyamine is such that about 50% to 100% of the available amine nitrogen positions are alkylated to tertiary amines. A preferred amount is from about 50% to about 80% alkylation of the amine nitrogen positions. Excess epihalohydrin beyond that required to fully alkylate all amine positions to tertiary amines is not preferred due to increased production of epihalohydrin by-products.

After complete addition of the polyamine, the temperature of the mixture is increased and/or the mixture is heated to crosslink and form the azetidinium salt. The rate of crosslinking is a function of concentration, temperature, agitation and addition conditions of the polyamine, all of which can be readily determined by one skilled in the art. The rate of crosslinking can be accelerated by adding small particles of polyamines or other polyamines of this invention or by adding various bases at or near the crosslinking temperature.

The resin can be stabilized against further crosslinking and gelation by addition of acid, dilution with water, or a combination of both. Usually suitably acidified to pH 5.0.

Preferred polyamines are bis-hexamethylenetriamine, hexamethylenediamine and mixtures thereof.

While not wishing to be bound by theory, it should be noted that resins such as Kymene® at high solids concentrations result in reduced bacterial growth due to increased viscosity and gelling of the resin, and loss of product functionality (= loss of functional groups) due to crosslinking, thus These resins are difficult and do not readily biodehalogenate at high solids concentrations, eg greater than 15% total solids. By controlling the conditions, including pH, time, temperature, and the concentration of microorganisms or enzymes, the biological dehalogenation of Daniels resin and tertiary amine resin can be achieved under the condition that higher total solids can be obtained. Preferred conditions for Daniels resins such as Kymene(R) 736 are 15-40 wt% total solids, preferably 18-30 wt%, even more preferably 18-22 wt%. Preferred conditions for tertiary amino-based resins are total solids content of 15-40 wt%, preferably 18-35 wt%, even more preferably 18-28 wt%. The preferred pH range for both Daniels resins and tertiary amine-based resins is pH 5.0-8.0, more preferably pH 5.5-7.5. The preferred temperature range for both Daniels resins and tertiary amine-based resins is 20-40°C, more preferably 25-35°C. Preferably the biodehalogenation step is completed within 48 hours or less from inoculation. More preferably the biodehalogenation step is completed within 24 hours or less from inoculation.

Unexpectedly, biodehalogenation can be performed at high solids concentrations due to lack of water for the microorganisms, high osmotic pressure at higher solids contents, and uncertain issues such as the concentration of low molecular weight species. Also, it is contemplated that pretreatment may be required to remove higher residues, such as by dilution or filtration. Furthermore, it was unexpected that biodehalogenation could be achieved within a reasonable time, eg within 48 hours. Also, storage instability at high solids concentrations should be expected; however, the resin compositions of the present invention are storage stable and are not prone to gelling. For high solids, the advantages of the present invention are obtained regardless of whether the resin composition has been treated to remove or reduce CPD forming species.

Also, in the present invention, the high solids, wet strength resin can be biodehalogenated. In addition, enzyme treatment may be performed simultaneously with biological dehalogenation treatment. Although prepolymer-based resins such as Kymene E7219 can undergo biodehalogenation or enzymatic treatment during biodehalogenation without endcapping, the preferred wet strength resins are WO 99/09252 and US6222006 (the contents of which are adopted by The capping resins described in incorporated herein by reference). While not wishing to be bound by theory, it should be noted that the capping agent is preferably not an inhibitor of biodehalogenation. For example, residual hexanoic acid from the production of end-capped prepolymers inhibits microbial biodehalogenation, whereas residual acetic acid does not inhibit microbial biodehalogenation. The preferred solids content of the wet strength resin is 15-40 wt%, preferably 16-35 wt%, even more preferably 18-28 wt%. Another range that can be used in the present invention is 15-30 wt%.

In order to describe the present invention more clearly, the following non-limiting examples are provided for illustration and are not construed as limiting the scope of the present invention. All parts and percentages in the examples are by weight unless otherwise indicated. In addition, ND in the Examples means "not detected".

Example

Unless otherwise stated, Brookfield viscosities are measured at 25°C using a Brookfield LVDV-II+ programmable viscometer. The procedure used is based on Operating Instructions, Manual No. M/97-164. The viscometer will measure viscosity only if the correct spindle and rpm are used for the viscosity of the sample. All CPD and DCP measurements are on a wet basis unless otherwise stated.

The synthesis of embodiment 1 high solid polyaminopolyamide resin

A 3-L round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, addition funnel and mechanical stirrer. To the pot was added 775.0 g of 49.6 wt% aqueous poly(adipate-co-diethylenetriamine) (available from Hercules Incorporated) and 505.3 g of water. The solution was heated to 25°C, and then 162.5 g of epichlorohydrin (Aldrich, 99%) were added over about 2 minutes. The temperature was increased to 40°C and maintained at this temperature. 2.45 hours after the addition of epichlorohydrin, 1046.5 g of water were added and the reaction mixture was heated. After the reaction mixture had reached 50°C (20 minutes), 7.54 g of 96% sulfuric acid were added. The temperature was raised to 70°C and its Gardner-Holdt viscosity at 25°C was checked. After its Gardner-Holdt viscosity reached G, the reaction was quenched by adding 187.5 g of water containing 12.90 g of 96% sulfuric acid. The reaction mixture was cooled to 25 °C. The pH was adjusted to pH 3.5 by adding 3.00 g of 96% sulfuric acid. The resin has 21.08% total solids and a Brookfield viscosity of 153 cps.

Example 2 Enzyme treatment of polyaminopolyamide-epi resin (embodiment 1)

A 250-mL round-bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, and mechanical stirrer. 200.0 g of Example 1 was added to the flask. The pH was raised to 7.58 with 4.88 g of 30% aqueous sodium hydroxide. A 5 g aliquot was removed, its pH was lowered to about 3 with 96% sulfuric acid and analyzed by GC. 5.18 g of ALCALASE Form DX 2.5L (available from Novozymes, used as received) was then added. The temperature was raised from 21°C to 30°C in 15 minutes and the Gardner-Holdt viscosity at 25°C was measured. A 5 g aliquot of the reaction mixture was removed and its pH was lowered to about 3 with 96% sulfuric acid at 1, 2, 4, 6 and 8 hours after the addition of ALCALASE and analyzed by GC. Its pH was adjusted to pH 7.5 with 0.27 g of 30% aqueous sodium hydroxide solution at 2 hours and with 0.18 g of 30% aqueous sodium hydroxide solution at 4 hours. After 8 hours, it was adjusted by adding 2.22 g of 96% sulfuric acid. The pH dropped to 3.4. The resin has a Brookfield viscosity (at 25°C) of 95 cps.

Example 3: The biological dehalogenation of Example 2

A 250-mL round bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, air sparge tube, and mechanical stirrer. 100.0 g of Example 2 and 55.56 g of water were added to the flask. The pH was raised to 5.9 with 2.24 g of 30% aqueous sodium hydroxide solution, and then 17.28 g of a mixture of microorganisms containing an inoculum derived from a biodehalogenated polyaminopolyamide-epichlorohydrin resin was added. This represents a starting value for a cell concentration of about 10 ⁵ to about 10 ⁶ cells/ml. This starting value corresponds to a final treatment level of about 10 ⁹ cells/ml as the process proceeds. The inoculum was added together with 1.36 g of nutrient solution. (The nutrient solution is made up of the tap water of the potassium dihydrogen phosphate of 8026ppm, the urea of 27480ppm, the magnesium sulfate of 4160ppm and the calcium chloride of 840ppm.) The microorganisms used are: Arthrobacter histidinophilus (HK1) and Agrobacterium radioactive ( HK7). Air sparging was started, its temperature was maintained at 30°C, and its pH was maintained at 5.8 by periodic addition of 30% aqueous sodium hydroxide solution. After 48 hours, the mixture was cooled to room temperature and its pH was adjusted to pH 3 with 0.97 g of 96% sulfuric acid .0 and 2.05 g of the biocide solution was added. [The biocide solution consisted of 10% active Proxel(R) BD (from Zeneca Biocides) and 1.67% potassium sorbate in deionized water. ] The resin has 14.5 wt% total solids and has a Brookfield viscosity (at 25°C) of 62 cps.

Acid test

The following acid test was used to assess the amount of CPD-generating material therein. Pour a portion of the resin to be assayed into a bottle containing a magnetic stirrer. Adjust its pH to pH 1.0 with 96% sulfuric acid. The vial was capped and placed in a 50°C water bath maintained at 50°C with stirring. Aliquots were periodically removed from the vials and analyzed by GC. The CPD produced after 24 hours was used to evaluate the amount of CPD producing substance. See Table 1 for the results.

Table 1 Resin information temperature(℃) time (hours) Gardner-Holdt viscosity Epi(ppm) 1,3-DCP (ppm) 2,3-DCP (ppm) 3-CPD (ppm) Example 2a twenty one 0 N 15 1746 1.3 276 Example 2b 30 1 FG 20 2004 1.6 478 Example 2c 30 2 EF twenty two 1720 1.7 508 Example 2d 30 4 DE twenty four 1802 1.4 680 Example 2e 30 6 DE 26 1753 1.5 664 Example 2f 30 8 EF ---- ---- ---- ---- Example 3 ---- ---- ---- 0.1 ND 0.8 ND acid test 50 twenty four ---- ND ND 0.10 0.66

   Comparative Example 4: Enzyme treatment of polyaminopolyamide-epi resin

Enzyme Treatment: A portion of Example 1 was diluted to 13.5% total solids. A 1-L round bottom flask was equipped with a condenser, pH meter, temperature controlled circulating bath and mechanical stirrer. 900.0 g of 13.5% Example 1 was added to the flask. The pH was raised to 7.54 with 13.85 g of 30% aqueous sodium hydroxide. A 5 g aliquot was removed, its pH lowered to about 3 with 96% sulfuric acid and analyzed by GC. 15.0 g of ALCALASE Form DX 2.5L (available from Novozymes, used as received) was then added. Its temperature was raised from 22°C to 35°C in 15 minutes, and the Gardner-Holdt viscosity at 25°C was measured. A 5 g aliquot of the reaction mixture was removed and its pH was lowered to about 3 with 96% sulfuric acid at 1, 2, 4, 6 and 8 hours after the addition of ALCALASE and analyzed by GC. Its pH was adjusted to pH 7.5 with 0.80 g of 30% aqueous sodium hydroxide solution at 2 hours, 0.48 g of 30% aqueous sodium hydroxide solution at 4 hours and 0.78 g of 30% aqueous sodium hydroxide solution at 6 hours. At 6 hours, the temperature was raised to 38°C. After 8 hours, the pH was lowered to 3.5 by adding 6.54 g of 96% sulfuric acid. The resin has a Brookfield viscosity (at 25°C) of 32 cps.

Biodehalogenation: A 1-L round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube and mechanical stirrer. Into the flask was added 700.0 g of the resin produced above. The pH was raised to 5.9 with 8.21 g of 30% aqueous sodium hydroxide solution, and then 77.8 g of a mixture containing microorganisms derived from the inoculum of the biodehalogenated polyaminopolyamide-epichlorohydrin resin was added. This represents a starting value for a cell concentration of about 10 ⁵ to about 10 ⁶ cells/ml. This starting value corresponds to a final treatment level of about 10 ⁹ cells/ml as the process proceeds. The inoculum was added together with 6.12 g of nutrient solution. (the nutrient solution is made up of the tap water of the potassium dihydrogen phosphate of 8026ppm, the urea of 27480ppm, the magnesium sulfate of 4160ppm and the calcium chloride of 840ppm.) The microorganisms used are: Arthrobacter histidinophilus (HK1) and Agrobacterium radioactive ( HK7). Air sparging was started, its temperature was maintained at 30°C, and its pH was maintained at 5.8 by periodic addition of 30% aqueous sodium hydroxide solution. After 48 hours, the mixture was cooled to room temperature and its pH was adjusted to pH 3 with 4.02 g of 96% sulfuric acid .0 and 8.42 g of biocide solution was added. [The biocide solution consisted of 10% active Proxel(R) BD (from Zeneca Biocides) and 1.67% potassium sorbate in deionized water. ] The resin has 14.77 wt% total solids and has a Brookfield viscosity (at 25°C) of 61 cps.

Example 5: Handsheet evaluation of Example 3 and Comparative Example 4

Handsheets were prepared on a Noble and Wood handsheet machine at pH 7.5 with 50:50 Rayonier bleached kraft: Crown Vantage bleached hardwood kraft wet and dry pulp boards refined to 500 mL Canadian Standard Freeness. Handsheets with 40 lb/3000 sq. ft. containing 0.5-1.0% treated resin (based on solids of untreated resin) were produced. The handsheets were wet pressed to 33% solids and dried on a can dryer at 230°C for 55 seconds to 3-5% moisture. Paper was conditioned and tested according to TAPPI Method T-402. Dry tensile strength was determined using TAPPI method T-494. Wet tensile strength was determined using TAPPI method T-456 with soaking for 2 hours. The CPD of paper products is determined by the following steps:

The steps of CPD in paper products

                                                     , 

Samples were cut and extracted with water at 23°C (±2°C) for 24 hours with occasional mixing. After the extraction time, the extract is filtered if necessary.

NOTE: Make sure all paper is soaked in water.

Steps

1. Put on protective gloves, cut the sample into small pieces (about 1cm×1cm), and collect them in plastic bags. Mix the pieces well.

2. Weigh 10g of the sample, to the nearest 0.0001g, and put it into the Erlenmeyer flask.

3. Add 200mL of reagent grade water and stopper the flask.

4. Place the flask in a water bath at 23°C (±2°C) for 24 hours.

5. Decant the solution into a 250mL volumetric flask. The preparation was filtered, if necessary, using a fritted glass filter funnel with a filter flask. The tablets were rinsed 2 times with additional reagent grade water and filled to the mark.

equipment

1. Erlenmeyer flask, wide neck, ground glass stopper

2. Volumetric flask, 250mL

3. Fritted glass filter funnel (available from Lab Glass as cat# 1G-7080-170), with filter flask, 500 mL

4. Keep the water bath at a constant temperature of 23°C (±2°C).

5. Paper cutter or scissors

6. Analytical balance, capable of weighing to the nearest 0.0001g.

Reagents

1. Water, available from Burdick & Jackson as cat. #365-4

ECD method - ether elution and derivatization

The analyte is separated from the aqueous extract by a liquid-liquid extraction column. DCP and 3-CPD were derivatized with heptafluorobutyrylimidazole (HFBI) and analyzed by gas chromatography using microelectron capture detector (μ-ECD).

Steps

1. Pipette 20 mL of the extracted aqueous solution into a 35-mL vial.

2. Add 2.34g NaCl to the vial and shake well until the NaCl dissolves.

3. Pour the solution onto an extrelut column and let stand for 15 minutes.

4. After waiting, elute with 250 mL of eluent (95% diethyl ether/isooctane). (collect the eluate in a volumetric flask)

5. Pour the eluate into a 500mL round bottom flask

6. Use a rotary evaporator to remove the solvent (note: the vacuum does not exceed 200mm Hg), until about 15mL remains.

7. Pipette 1 mL of internal standard solution into the remaining isooctane.

8. A method blank of reagent grade water prepared according to steps 2-7 must also be performed to detect interferences.

Derived

1. Add 200 μL of HFBI to the flask using a syringe or micropipette. Stopper the flask and swirl the solution to mix well.

2. Let the flask stand at room temperature for 15 minutes.

3. Quantitatively transfer the solution to a 25 mL mixing cylinder and fill to the mark with isooctane.

4. Add about 1.5mL of reagent grade water to the volumetric flask, stopper and shake well to mix. A precipitate formed but disappeared when mixed well with water.

5. After phase separation, remove approximately 20 mL of the organic phase and place in a 30 mL glass vial containing 2 mL of reagent grade water. Shake vigorously for 1 min.

6. After phase separation, the aqueous layer was removed and discarded. The organic phase was analyzed by gas chromatography using a micro-electron capture detector (ECD).

Reagents

1. Diethyl ether, available as Cat. No. 31690 from FLUKA, P.O. Box 355, Milwaukee, WI.

^** Analytically pure quality must be used; the ether can be neither dried nor stabilized with ethanol.

1. Water, available from Burdick & Jackson as cat. #365-4

2. Sodium chloride

3. 1,3-DCP; available from TCI Americas as Cat. No. D0402.

4. 3-CPD; available from Aldrich as Cat. No. 10227-1.

5. Acetonitrile, nanoscale, available from Fisher as Cat. No. 2442.

6. Isooctane, EM Science Cat.No.TX1389

7. Eluent: 95mL diethyl ether/5mL isooctane.

8. Heptafluorobutyrylimidazole (HFBI), available from Pierce as Cat. No. 44211

9. 3-methoxy-1,2-propanediol (internal standard)

10. Solid phase extraction column, Supelco, Supelco Park, Bellefonte, PA 16823-0048, prepared according to Section XXXCat.No.57022.

11. Varian Hydromatrix; available from Varian, Inc. as Cat. No. 00198003.

equipment

1. Gas Chromatograph, Hewlett Packard Model 5890, or equivalent, capable of linear column temperature programming and equipped with a Micro-Electron Capture Detector (μ-ECD).

2. Data processing system, Hewlett Packard ChemStation or equivalent.

3. Chromatographic column, DB-5MS, 60m×0.25mm I.D.—available as Cat.No.122-5562 from J&W Scientific Inc., 91 Blur Ravin Road, Folsom, CA 95630.

4. Flask, capacity, glass stopper, 5mL, 10mL, 25mL, 50mL, 100mL, 250mL.

5. Vials, glass with Teflon-lined screw caps, 17 mL, 30 mL, 4 oz.

6. Pipette, transfer 0.5, 1, 2, 3, 5, 10, 20mL Class A.

7. Drug stopper, glass-Fisher, Cat.No.13-701

8. Analytical balance capable of weighing to the nearest 0.0001g.

9. Solid phase extraction column, Supelco, Supelco Park, Bellefonte, PA 16823-0048, prepared according to Section XXX. Cat. No. 57022.

10. Glass wool

11. 500 mL round bottom flasks with stoppers available from Lab Glass as Cat. No. 013 and Cat. No. 114.

12. Rotary vacuum evaporator, operated at 35-40°C/800mbar

13. 500μL syringe or disposable micropipette

14. Mixing cylinder Type A, 25 mL; available from Fisher as Cat. No. 08-563-1F.

Internal standard solution (low level)

1. Weigh 50 mg of 3-methoxy-1,2-propanediol into a 50-mL volumetric flask and record the weight to the nearest 0.0001 g.

2. Dilute to volume with acetonitrile.

3. Pipette 0.25 mL of the solution from step 2 into a 100-mL volumetric flask and dilute to volume with diethyl ether.

4. Pipette 10.0 mL of the solution in step 3 into a 100 mL volumetric flask and dilute to volume with diethyl ether.

              1, 3-DCP, 3-CPD calibration solution (low level)

1. Weigh 50 mg of 1,3-dichloro-2-propanol into a 50-mL volumetric flask and record the weight to the nearest 0.0001 g.

2. Dilute to volume with acetonitrile.

3. Pipette 0.5 mL of the solution from step 2 into a 10-mL volumetric flask and dilute to volume with diethyl ether.

4. Weigh 50 mg of 3-chloro-1,2-propanediol into a 50-mL volumetric flask and record the weight to the nearest 0.0001 g.

5. Dilute to volume with acetonitrile.

6. Pipette 0.5 mL of the solution from step 5 into a 10-mL volumetric flask and dilute to volume with diethyl ether.

7. Combine the solutions from Step 3 and Step 6 into a 30-mL vial and mix well

8. Pipette 2.5 mL of the solution in step 7 into a 100 mL volumetric flask and dilute to volume with diethyl ether.

9. Pipette 10.0 mL of the solution from step 8 into a 100-mL volumetric flask and dilute to volume with diethyl ether. This is the calibration stock solution.

Calibration curve (low level):

1. Pipette 0.1 mL of the calibration stock solution into a 25-mL volumetric flask containing 1.0 mL of the internal standard solution. Using a pipette, add 5.9 mL of diethyl ether to the flask. This is Calibration Level #1.

2. Pipette 0.2 mL of the calibration stock solution into a 25-mL volumetric flask containing 1.0 mL of the internal standard solution. Using a pipette, add 5.8 mL of diethyl ether to the flask. This is calibration level #2.

3. Pipette 0.5 mL of the calibration stock solution into a 25-mL volumetric flask containing 1.0 mL of the internal standard solution. Using a pipette, add 5.5 mL of diethyl ether to the flask. This is calibration level #3.

4. Pipette 1.0 mL of the calibration stock solution into a 25-mL volumetric flask containing 1.0 mL of the internal standard solution. Using a pipette, add 5.0 mL of diethyl ether to the flask. This is calibration level #4.

5. Pipette 2.0 mL of the calibration stock solution into a 25-mL volumetric flask containing 1.0 mL of the internal standard solution. Using a pipette, add 4.0 mL of diethyl ether to the flask. This is calibration level #5.

6. Add 15 mL of isooctane to each volumetric flask in steps 1-6.

7. Using a syringe, add 200 μL of HFBI to each volumetric flask from step 7, then stopper and let stand at room temperature for 15 minutes with occasional shaking.

8. Dilute each flask with isooctane to a final volume of 25 mL.

9. Add approximately 1.5 mL of reagent grade water to each volumetric flask, stopper and shake to mix thoroughly. A precipitate will form but will disappear when mixed well with water.

10. After phase separation, transfer approximately 20 mL of the organic phase to each 30-mL glass vial containing 2 mL of reagent grade water. Shake vigorously for 1 min.

11. After phase separation, the aqueous layer was removed and discarded. The organic phase was analyzed by gas chromatography using a micro-electron capture detector (μ-ECD) to determine a calibration curve.

GC operating conditions

temperature

column

Initially 50℃

Initial hold time 2 minutes

  Initial Speed                                        

                                    

                                                   

                                                                   

Finally 300℃

  Final Hold Time                                                

Entrance 250℃

 Detector temperature 320°C

flow rate

Helium (carrier gas) 1.5mL/min at 20psi (column head at 35°C)

Argon/methane 60mL/min

SECTION XXX

Preparing EXTRELUT QE Columns

1. Using a solid phase extraction reservoir, press about 0.5g of glass wool to the bottom.

2. Weigh 18g of Varian Hydromatrix and pour into the reservoir. Using a glass probe, wrap the extrelut tightly.

3. Put about 0.5g of glass wool on top of the reservoir.

The results are reported in Table 2. The data show that the enzyme treatment at 21% solids results essentially the same as the treatment at 13.5% solids. These results make the enzyme treatment more economical.

Table 2: Natural aging paper results

natural aging paper Normalized Basis Weight Example % added resin Dry Tensile Strength (lbs/in) Wet Tensile Strength (lbs/in) % wet/dry % of comparative example XX CPD in paper (ppb) blank ---- 17.55 0.53 3 ---- <3 Comparative Example 4a 0.25 24.47 3.89 16 ---- <3 Comparative Example 4b 0.50 24.61 4.86 20 ---- <3 Comparative Example 4c 1.00 24.65 5.70 twenty three ---- 10 Example 3a 0.25 22.58 3.60 16 93 <3 Example 3b 0.50 23.88 4.63 19 95 <3 Example 3c 1.00 24.72 5.38 twenty two 94 7

Example 6-17: General steps for enzymatic treatment of polyaminopolyamide-epi resin

A 500-mL round-bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, and mechanical stirrer. To the flask was added 400.0 g of Kymene(R) E7219 (available from Hercules Incorporated, Wilmington, DE; 21.51% solids, Brookfield viscosity 267 cps at 25°C). Its pH was raised with 30% aqueous sodium hydroxide. A 5 g aliquot was removed, its pH was lowered to about 3 with 96% sulfuric acid and analyzed by GC. ALCALASE 2.5L Form DX (available from Novozymes, used as received) was added (in amounts indicated in Table 3). The temperature was raised to the desired processing temperature within 15 minutes and the Gardner-Holdt viscosity at 25°C was checked. A 5 g aliquot of the reaction mixture was removed and its pH was lowered to about 3 with 96% sulfuric acid at 1, 2, 4, 6 and 8 hours after the addition of ALCALASE and analyzed by GC. The pH was checked hourly and adjusted with 30% aqueous sodium hydroxide if the pH shifted by more than 0.10. After 8 hours, the pH was lowered to 3.5 by adding 96% sulfuric acid. If the Gardner-Holdt viscosity reading is between two letters, record both letters in the table. If the viscosity increases more than desired, stop adjusting its pH with 30% aqueous sodium hydroxide. If its viscosity increases to the point where it is dangerously gelled, its pH is lowered to 3.5 by adding 96% sulfuric acid. BV (cps) is the Brookfield viscosity (measured at 25°C) of the final resin. The ALCALASE:active solids ratio is defined as the amount of ALCALASE 2.5L Form DX (used as is) compared to the amount of active solids in the resin. See Table 3 for details.

Example 18-19: General steps for enzymatic treatment of polyaminopolyamide-epi resin

A 250-mL round bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, and mechanical stirrer. 200.0 g of Example 1 was added to the flask. Its pH was raised with 30% aqueous sodium hydroxide. A 4g aliquot was removed, its pH lowered to about 3 with 96% sulfuric acid and analyzed by GC. ALCALASE 2.5L Form DX (available from Novozymes, used as received) was added (in amounts indicated in Table 3). The temperature was raised to the desired processing temperature within 15 minutes and the Gardner-Holdt viscosity at 25°C was checked. A 4 g aliquot of the reaction mixture was removed and at 1, 2, 4, 6 and 8 hours after the addition of ALCALASE, if no gelling, its pH was lowered to about 3 with 96% sulfuric acid and analyzed by GC. The pH was checked hourly and adjusted with 30% aqueous sodium hydroxide if the pH shifted by more than 0.10. After 8 hours, the pH was lowered to 3.5 by adding 96% sulfuric acid. If the Gardner-Holdt viscosity reading is between two letters, record both letters in Table 3. If the viscosity increases more than desired, stop adjusting its pH with 30% aqueous sodium hydroxide. With these two reactions, its viscosity increases to the gel point. BV (cps) is the Brookfield viscosity (measured at 25°C) of the final resin. The ALCALASE:active solids ratio is defined as the amount of ALCALASE 2.5L Form DX (used as is) compared to the amount of active solids in the resin. See Table 3 for details.

table 3 Resin Example ALCALASE: active solid pH temperature(℃) time (hours) Gardner-Holdt viscosity BV(cps) Example 6a 1.0:8.3 7.2 twenty one 0 IJ Example 6b 1.0:8.3 7.2 30 1 EF Example 6c 1.0:8.3 7.2 30 2 DE Example 6d 1.0:8.3 7.2 30 4 D. Example 6e 1.0:8.3 7.2 30 6 D. Example 6f 1.0:8.3 7.2 30 8 D. 84 Example 7a 1.0:8.3 7.8 twenty one 0 IJ Example 7b 1.0:8.3 7.8 30 1 EF Example 7c 1.0:8.3 7.8 30 2 EF Example 7d 1.0:8.3 7.8 30 4 EF Example 7e 1.0:8.3 7.8 30 6 FG Example 7f 1.0:8.3 7.8 30 8 HI 255 Example 8a 1.0:16.6 7.2 twenty two 0 IJ Example 8b 1.0:16.6 7.2 25 1 GH Example 8c 1.0:16.6 7.2 25 2 G Example 8d 1.0:16.6 7.2 25 4 f Example 8e 1.0:16.6 7.2 25 6 EF Example 8f 1.0:16.6 7.2 25 8 EF 105 Example 9a 1.0:16.6 7.8 twenty two 0 IJ Example 9b 1.0:16.6 7.8 25 1 GH Example 9c 1.0:16.6 7.8 25 2 GH Example 9d 1.0:16.6 7.8 25 4 I Example 9e 1.0:16.6 7.8 25 6 JK Example 9f 1.0:16.6 7.8 25 7 LM 379 Example 10a 1.0:11.1 7.2 twenty three 0 IJ Example 10b 1.0:11.1 7.2 25 1 FG Example 10c 1.0:11.1 7.2 25 2 f Example 10d 1.0:11.1 7.2 25 4 E. Example 10e 1.0:11.1 7.2 25 6 DE Example 10f 1.0:11.1 7.2 25 8 D. 77 Example 11a 1.0:11.1 7.8 twenty three 0 IJ Example 11b 1.0:11.1 7.8 25 1 FG Example 11c 1.0:11.1 7.8 25 2 f Example 11d 1.0:11.1 7.8 25 4 EF Example 11e 1.0:11.1 7.8 25 6 f Example 11f 1.0:11.1 7.8 25 8 f 149 Example 12a 1.0:8.3 7.2 twenty two 0 HI Example 12b 1.0:8.3 7.2 25 1 FG Example 12c 1.0:8.3 7.2 25 2 EF Example 12d 1.0:8.3 7.2 25 4 DE Example 12e 1.0:8.3 7.2 25 6 C Example 12f 1.0:8.3 7.2 25 8 BC 60 Example 13a 1.0:8.3 7.8 twenty two 0 HI Example 13b 1.0:8.3 7.8 25 1 FG Example 13c 1.0:8.3 7.8 25 2 EF Example 13d 1.0:8.3 7.8 25 4 DE Example 13e 1.0:8.3 7.8 25 6 C Example 13f 1.0:8.3 7.8 25 8 cd 85 Example 14a 1.0:8.3 7.2 twenty two 0 HI Example 14b 1.0:8.3 7.2 35 1 FG Example 14c 1.0:8.3 7.2 35 2 EF Example 14d 1.0:8.3 7.0 35 4 f Example 14e 1.0:8.3 6.9 35 6 FG Example 14f 1.0:8.3 6.8 35 8 GH 175 Example 15a 1.0:8.3 7.5 twenty two 0 HI Example 15b 1.0:8.3 7.5 35 1 FG Example 15c 1.0:8.3 7.5 35 2 f Example 15d 1.0:8.3 7.3 35 4 JK Example 15e 1.0:8.3 7.3 35 4.5 N 434 Example 16a 1.0:16.6 7.5 twenty two 0 HI Example 16b 1.0:16.6 7.5 25 1 G Example 16c 1.0:16.6 7.5 25 2 G Example 16d 1.0:16.6 7.5 25 4 FG Example 16e 1.0:16.6 7.5 25 6 EF Example 16f 1.0:16.6 7.5 25 8 EF 138 Example 17a 1.0:11.1 7.5 twenty two 0 HI Example 17b 1.0:11.1 7.5 25 1 FG Example 17c 1.0:11.1 7.5 25 2 EF Example 17d 1.0:11.1 7.5 25 4 DE Example 17e 1.0:11.1 7.5 25 6 D. Example 17f 1.0:11.1 7.5 25 8 D. 80 Example 18a 1.0:16.3 7.5 twenty two 0 h Example 18b 1.0:16.3 7.5 33 1 I Example 18c 1.0:16.3 7.5 33 2 K Example 18d 1.0:16.3 7.5 33 3 V Example 18e 1.0:16.3 7.5 33 4 WX Example 19a 1.0:8.1 7.5 twenty two 0 h Example 19b 1.0:8.1 7.5 33 1 f Example 19c 1.0:8.1 7.5 33 2 EF Example 19d 1.0:8.1 7.5 33 4 f Example 19e 1.0:8.1 7.5 33 6 h Example 19f 1.0:8.1 7.5 33 7 L Example 19g 1.0:8.1 7.5 33 8 gel

Example 20 Polyaminopolyamide-epi Resin

  Combined enzymatic treatment and biological dehalogenation

Scale up by 1 (to prepare starter)

A portion of Kymene(R) E7219 (available from Hercules Incorporated, Wilmington, DE; 21.51% solids, Brookfield viscosity 267 cps at 25°C) was diluted to 13.5% total solids. A 400-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 400 g of the 13.5% Kymene(R) E7219 was added to the flask. The pH was raised to 7.54 with 7.42 g of 30% aqueous sodium hydroxide. A 5 g aliquot was removed, its pH lowered to about 3 with 96% sulfuric acid and analyzed by GC. Then 3.33 g of ALCALASE 2.5L type DX (available from Novozymes, used as received) were added, followed by 44.4 g of a mixture of microorganisms containing the inoculum from the biodehalogenated polyaminopolyamide-epichlorohydrin resin. This represents a starting value for a cell concentration of about 10 ⁵ to about 10 ⁶ cells/ml. This starting value corresponds to a final treatment level of about 10 ⁹ cells/ml as the process proceeds. The inoculum was added together with 3.50 g of nutrient solution. (the nutrient solution is made up of the tap water of the potassium dihydrogen phosphate of 8026ppm, the urea of 27480ppm, the magnesium sulfate of 4160ppm and the calcium chloride of 840ppm.) The microorganisms used are: Arthrobacter histidinophilus (HK1) and Agrobacterium radioactive ( HK7). Air sparging was started and the temperature was maintained at 30°C. The treatment was monitored by Gardner-Holdt viscosity and bacterial growth by optical density ( _OD600 ). _OD600 was determined by measuring its optical density at a wavelength of 600 nm using a SpectronicGenesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length . Periodically, 5 g aliquots were withdrawn, their pH was lowered to about 3 with 96% sulfuric acid and analyzed by GC. The pH of the treatment was maintained at 7.1-7.5 during the first 30 hours by the periodic addition of 30% aqueous sodium hydroxide. After 30 hours, the pH was lowered to 5.8 by the addition of 96% sulfuric acid. After 48 hours, the resulting mixture was used as Scale up the inoculum for 2.

Scale up 2

A portion of Kymene(R) E7219 (available from Hercules Incorporated, Wilmington, DE; 21.51% solids, Brookfield viscosity 267 cps at 25°C) was diluted to 13.5% total solids. A 2-L round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube and mechanical stirrer. 1600 g of the 13.5% Kymene(R) E7219 was added to the flask. The pH was raised to 7.52 with 30.38 g of 30% aqueous sodium hydroxide. A 5 g aliquot was removed, its pH lowered to about 3 with 96% sulfuric acid and analyzed by GC. 13.32 g of ALCALASE 2.5L type DX (available from Novozymes, used as received) was then added, followed by 177.8 g of the inoculum from Scale-up 1 above, along with 14.0 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. The treatment was monitored by Gardner-Holdt viscosity and bacterial growth by optical density ( _OD600 ). _OD600 was determined by measuring its optical density at a wavelength of 600 nm using a Spectronic Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length . Periodically, 5 g aliquots were withdrawn, their pH was lowered to about 3 with 96% sulfuric acid and analyzed by GC. The pH of the treatment was maintained at 7.2-7.5 during the first 8.5 hours by periodic addition of 30% aqueous sodium hydroxide. During the remaining processing time, its pH was maintained at pH 6.8-7.2 by periodic addition of 30% aqueous sodium hydroxide solution. After 48 hours, the mixture was cooled to room temperature and its pH was adjusted to pH 2.0 with 12.80 g of 96% sulfuric acid. 8, and add 19.26g of biocide solution. [The biocide solution consisted of 10% active Proxel(R) BD (from Zeneca Biocides) and 1.67% potassium sorbate in deionized water. ] The results of the detection process are shown in Table 4.

Table 4 Aliquot time pH(30℃) Gardner viscosity _OD600 (absolute value) DCP (ppm) CPD(ppm) -1 "0" 7.49(21℃) D/E 0.058 679 204 -78A 0 Time "0" is just after addition of NaOH, time 0 is later after addition of Alcalase. ---- 0.25 7.43 ---- ---- ---- ---- -2 1 7.39 B/C ---- 589 242 -3 2 7.32 B/C 0.062 585 268 -4 4 7.32 B/C 0.067 586 309 -5 6 7.25 B 0.064 584 322 ---- 7 7.20-7.52 ---- ---- ---- ---- -6 8 7.50 B 0.061 554 331 -7 10 7.40 B 0.064 540 363 -8 14 7.26-7.45 B 0.064 519 399 -9 twenty four 7.20 D. 0.109 476 384 ---- 28 7.09 E/F 0.165 ---- ---- -10 30 7.05-5.79 G/H 0.201 422 296 ---- 32 5.83 h 0.260 ---- ---- ---- 34 5.84 ---- 0.300 ---- ---- -11 37 5.81 H/I 0.328 360 317 -12 48 5.60 I/J 0.473 ND 198 Aliquot time pH(30℃) Gardner viscosity _OD600 (absolute value) DCP (ppm) CPD(ppm) -1 "0" 7.48(26℃) D/E 0.071 745 256 -82A&-84B 0 Time "0" is just after addition of NaOH, time 0 is later after addition of Alcalase. ---- 0.25 7.46 ---- ---- -2 1 7.39 C/D ---- 639 394 -3 2 7.32 C/D 0.086 614 427 -4 4 7.20-7.40 C 0.108 579 531 -5 6 7.31 C 0.138 537 540 -6 8.5 7.16 B/C 0.198 391 629 -7 12.5 7.02 B/C 0.271 66 796 -8 twenty two 6.81 D. 0.481 ND 144 ---- twenty four 6.78-7.12 ---- ---- ---- The resin is light brownish yellow. ---- 28 7.02 D/E 0.560 -9 30 6.99 D/E 0.578 0.3 7.8 -10 48 6.95 D/E 0.611 0.3 0.5 acid test ---- ---- ---- ---- ND 1.1

Example 21:

Combined enzymatic treatment and biological dehalogenation of polyaminopolyamide-epi resins

(use 2 times the Alcalase in Example 20)

Scale up by 1 (to prepare starter)

A portion of Kymene(R) E7219 (available from Hercules Incorporated, Wilmington, DE; 21.51% solids, Brookfield viscosity 267 cps at 25°C) was diluted to 13.5% total solids. A 400-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 400 g of the 13.5% Kymene(R) E7219 was added to the flask. The pH was raised to 7.52 with 7.38 g of 30% aqueous sodium hydroxide. A 5 g aliquot was removed, its pH lowered to about 3 with 96% sulfuric acid and analyzed by GC. 6.66 g of ALCALASE 2.5L type DX (available from Novozymes, used as received) were then added, followed by 44.4 g of a mixture of microorganisms containing an inoculum derived from a biodehalogenated polyaminopolyamide-epichlorohydrin resin. This represents a starting value for a cell concentration of about 10 ⁵ to about 10 ⁶ cells/ml. This starting value corresponds to a final treatment level of about 10 ⁹ cells/ml as the process proceeds. The inoculum was added together with 3.50 g of nutrient solution. (The nutrient solution is made up of the tap water of the potassium dihydrogen phosphate of 8026ppm, the urea of 27480ppm, the magnesium sulfate of 4160ppm and the calcium chloride of 840ppm.) The microorganisms used are: Arthrobacter histidinophilus (HK1) and Agrobacterium radioactive ( HK7). Air sparging was started and the temperature was maintained at 30°C. The treatment was monitored by Gardner-Holdt viscosity and bacterial growth by optical density ( _OD600 ). _OD600 was determined by measuring its optical density at a wavelength of 600 nm using a SpectronicGenesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length . Periodically, 5 g aliquots were withdrawn, their pH was lowered to about 3 with 96% sulfuric acid and analyzed by GC. The pH of the treatment was maintained at 7.2-7.5 during the first 24 hours by periodic addition of 30% aqueous sodium hydroxide solution. After 24 hours, the pH shifted down to pH 6.71 over the course of 24 hours. After 48 hours, the Brookfield Viscosity was 71 cps (measured at 25°C). This mixture was used as the inoculum for Scale-up 2 below.

Scale up 2

A portion of Kymene(R) E7219 (available from Hercules Incorporated, Wilmington, DE; 21.51% solids, Brookfield viscosity 267 cps at 25°C) was diluted to 13.5% total solids. A 2-L round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube and mechanical stirrer. 1600 g of the 13.5% Kymene(R) E7219 was added to the flask. The pH was raised to 7.55 with 29.99 g of 30% aqueous sodium hydroxide. A 5 g aliquot was removed, its pH lowered to about 3 with 96% sulfuric acid and analyzed by GC. 26.64 g of ALCALASE 2.5L type DX (available from Novozymes, used as received) was then added, followed by 177.8 g of the inoculum from Scale-up 1 above, along with 14.0 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. The treatment was monitored by Gardner-Holdt viscosity and bacterial growth by optical density ( _OD600 ). _OD600 was determined by measuring its optical density at a wavelength of 600 nm using a Spectronic Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length . Periodically, 5 g aliquots were withdrawn, their pH was lowered to about 3 with 96% sulfuric acid and analyzed by GC. The pH of the treatment was maintained at 7.1-7.5 during the first 8 hours by periodic addition of 30% aqueous sodium hydroxide. During the remaining processing time, its pH was maintained at pH 6.8-7.2 by periodic addition of 30% aqueous sodium hydroxide solution. After 48 hours, the mixture was cooled to room temperature and its pH was adjusted to pH 2.0 with 12.85 g of 96% sulfuric acid. 8 and added 19.26 g of biocide solution. [The biocide solution consisted of 10% active Proxel(R) BD (from Zeneca Biocides) and 1.67% potassium sorbate in deionized water. ] The Brookfield viscosity of the resin was 30 cps (measured at 25°C). See Table 5 for the results of the detection process.

table 5 sample time pH(30℃) G/H viscosity _OD600 (absolute value) DCP (ppm) CPD(ppm) -1 "0" 7.50(21℃) D/E 0.060 770 269 -80B 0 Time "0" is just after addition of NaOH, time 0 is later after addition of Alcalase. ---- 0.25 7.44 ---- ---- ---- ---- -2 1 7.37 B ---- 657 412 -3 2 7.34 B 0.058 645 477 -4 4 7.30 A/B 0.063 677 526 -5 6 7.25 A/B 0.062 623 508 ---- 7 7.20-7.52 ---- ---- ---- ---- -6 8 7.50 A 0.059 599 504 -7 10 7.40 A/A-1 0.062 615 559 -8 14 7.26-7.47 A/A-1 0.065 592 569 -9 twenty four 7.22 A/B 0.139 516 560 ---- 28 7.12 A/B 0.259 ---- ---- -10 30 7.11 B 0.295 387 508 ---- 32 7.07 B 0.336 ---- ---- ---- 34 7.03 ---- 0.391 ---- ---- -11 37 6.93 B 0.475 ND 497 -12 48 6.71 C 0.716 ND ND

Scale up 2 Aliquot time pH(30℃) G/H viscosity _OD600 (absolute value) DCP (ppm) CPD(ppm) -1 "0" 7.48(26℃) D/E 0.101 778 237 -84B 0 Time "0" is just after addition of NaOH, time 0 is later after addition of Alcalase. ---- 0.25 7.46 ---- ---- -2 1 7.39 C ---- 641 384 -3 2 7.31 B/C 0.129 583 433 -4 4 7.10-7.40 B 0.199 507 531 -5 6 7.34 B 0.270 371 562 -6 8.5 ---- A/B 0.422 96 653 -7 12.5 6.75-7.14 A 0.618 ND 303 -8 twenty two 6.85 A/B 0.877 ND 0.5 ---- twenty four 6.85-7.09 ---- ---- ---- ---- The resin is dark orange, light tan. ---- 28 7.01-7.10 A/B 0.932 -9 30 7.08 A/B 0.959 ND ND -10 48 6.84 B 1.080 ND ND acid test ---- ---- ---- ---- ND 0.1

Examples 20 and 21 clearly show that enzyme treatment and biological dehalogenation treatment can be effectively combined. When using 2 times the Alcalase, the preferred equilibrium conditions give the preferred viscosity.

Example 22

Alcalase-biodehalogenation of Kymene® E7219

(See Table 6 for data and details)

Kymene(R) E7219 (available from Hercules Incorporated, Wilmington, DE; Zwijindrecht, Netherlands plant) was diluted to 13.40% and had a Brookfield viscosity of 76 cps.

Pasteurization: A 3-L round bottom flask was equipped with a condenser, temperature controlled circulating bath and mechanical stirrer. 2800 g of resin was added to the flask. Its pH was adjusted from 3.4 to pH 3.0 with concentrated sulfuric acid and heated from 25°C to 80°C within 15 minutes. The resin was held at 80°C for 15 minutes, cooled to 75°C within 10 minutes, then to 30°C. The pasteurized resin had a Brookfield viscosity of 48 cps and was stored in sterile containers.

Sterilization of Kymene E7219: Dilute a 500g portion of Kymene E7219 to 8%, put in an autoclavable bottle, and heat in an autoclave at 121°C for 20 minutes. The resin was cooled and used to start the scale-up 1 preparation of the resin inoculum. NOTE: Pasteurized Kymene E7219 (using the above conditions) was also successfully used to start the scale-up 1 preparation of the resin inoculum.

Biodehalogenation: Preparation of resin inoculum [Scale-up 1 (SU1)]: A 250-mL round bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, air sparge tube, and mechanical stirrer. Add 200 g of sterilized KymeneE7219 in the flask, and raise its pH to 7.2 with 3.28 g of 30% aqueous sodium hydroxide solution, then add 400 microliters of HK7 concentrated starter culture (add HK7 at 1:500 into the resin) [see Example 24 for the preparation of a concentrated starter culture] and add 1.75 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a ^Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. The pH of the reaction mixture was maintained by periodic addition of 30% aqueous sodium hydroxide. After 34 hours, 68 microliters of HK1 concentrated starter culture (HK1 added to resin at 1:3000) was added [see Example 24 for the preparation of concentrated starter culture]. After 43 hours, the resulting resin was used as an inoculum for SU2.

Scale up 2 (SU2)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 350 g of the pasteurized resin was added to the flask. Its pH was raised to 7.5 with 7.40 g of 30% aqueous sodium hydroxide solution, and then 5.03 g of ALCALASE 2.5L type DX (available from Novozymes), 87.5 g of SU1 resin inoculum (20% inoculum rate) and 3.06 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a ^Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. The pH of the reaction mixture was maintained by periodic addition of 30% aqueous sodium hydroxide. After 23 hours, the resulting resin was used as an inoculum for SU3. To increase the molecular weight of the resin (shown by Gardner-Holdt viscosity or Brookfield viscosity), a 200 g portion of the remaining resin (Brookfield viscosity 10 cps) was raised to pH 8.5 with 1.17 g of 30% aqueous sodium hydroxide solution and the temperature was increased to 40°C. After 3 hours, the resin had the desired viscosity and the reaction was quenched to pH 2.7 by the addition of concentrated sulfuric acid. The resulting resin had a Brookfield viscosity of 25 cps.

General steps for scaling up 3 (SU3) and repeating batch mode:

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 350 g of the pasteurized resin was added to the flask. Its pH was raised to 7.6 with 8.59 g of 30% aqueous sodium hydroxide solution, and then 4.38 g of ALCALASE 2.5L type DX (available from Novozymes), 87.5 g of SU2 resin inoculum (20% inoculum rate) and 3.06 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a ^Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. The pH of the reaction mixture was maintained by periodic addition of 30% aqueous sodium hydroxide. After 23 hours, the resulting resin was used as an inoculum for SU4. Concentrated sulfuric acid adjusted the pH of the remaining resin to pH 2.8, and 300 ppm potassium sorbate was added as a 10% aqueous solution. The resin has a Brookfield viscosity of 49 cps.

Scale-up 4 (SU4) batches: Procedure similar to SU3, see Table 6 for data and details.

Scale up to 5-10 batches: The procedure was similar to SU3, except that 13.40% Kymene E7219 was used without pasteurization (see Table 6 for data and details).

SU3-SU8 is a series of similar trials with 10% inoculum yielding successful and efficient batch biodehalogenation.

Table 6 Scale-up 1: 200 g 8% E7219 (autoclaved), no Alcalase, 400 μl HK7, 1.75 g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.25 ---- ---- 3.28 ---- ---- X33031-15-1 1 7.24 ---- 0.174 ---- 445 183 X33031-15-2 4 7.22 ---- 0.154 ---- 402 211 X33031-15-3 20 7.15 ---- 0.138 ---- 259 309 X33031-15-4 twenty four 7.12 ---- 0.152 ---- 222 333 X33031-15-5 28 7.07-7.27 ---- 0.177 0.12 140 375 ---- 32 7.17 ---- 0.211 ---- ---- ---- ---- 34 7.13 ---- 0.249 Add 68 µl of HK1 inoculum ---- ---- 7.13 ---- 0.272 ---- ---- ---- X33031-15-6 43 6.84 ---- 0.657 ---- ND 0.36 Scale-up 2: 350g 13.5% E7219 (pasteurized), 5.03g Alcalase, 87.5g-15, 3.06g nutrient solution sample time (hours) (pH 7.50)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.49 ---- ---- 7.40 ---- ---- X33031-18-1 1 7.39-7.64 AB 0.128 0.65 434 485 X33031-18-2 4 7.41-7.64 A 0.170 0.49 10 814 X33031-18-3 7 7.50-7.6 A 0.257 0.26 ND 720 X33031-18-4 10 7.44-7.64 A 0.372 0.29 ND 380 X33031-18-5 13 7.47-7.59 A 0.543 0.22 ND 0.56 X33031-18-6 twenty three 7.56 A-1/A 0.678 ---- ND 0.38 The inoculum was removed for the next batch and the remainder was set aside for crosslinking. With a Brookfield viscosity of 10 cps (at pH 7.50), 200 g of -18 starts to crosslink after 8 hours: 7.20(24℃) ---- 7.11(31℃) ---- 0 8.50(31℃) ---- 1.17 0.5 8.23(40℃) 1 8.01 1.5 7.93 A-1/A 2 7.82 2.5 7.74 X33047-27-1 3 7.68 Stop the reaction with sulfuric acid 2.65

The final resin has a Brookfield viscosity of 25 cps. Scale-up 3: 350g 13.5% E7219 (pasteurized), 4.38g Alcalase, 87.5g-18, 3.06g nutrient solution sample time (hours) (pH7.60)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.63 ---- ---- 8.59 ---- ---- X33031-20-1 1 7.51-7.79 AB 0.148 0.70 476 430 X33031-20-2 4 7.54-7.73 A 0.209 0.40 186 616 X33031-20-3 7 7.49-7.74 A 0.307 0.56 ND 715 X33031-20-4 10 7.52-7.71 A 0.436 0.37 ND 437 X33031-20-5 13 7.49-7.70 A 0.594 0.37 ND 152 X33031-20-6 twenty three 7.48 BC 0.726 ---- ND 0.29

The final resin had a Brookfield viscosity of 49 cps. Scale-up 4: 350g 13.5% E7219 (pasteurized), 4.38g Alcalase, 87.5g-20, 3.06g nutrient solution sample time (hours) (pH 7.50)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.60 ---- ---- 7.51 ---- ---- X33031-23-1 1 7.53-7.62 AB 0.176 0.24 588 360 X33031-23-2 4 7.40-7.61 AB 0.211 0.48 516 470 X33031-23-3 7 7.46-7.63 A 0.258 0.38 227 736 X33031-23-4 10 7.47-7.62 A 0.312 0.29 ND 811 X33031-23-5 12.5 7.52-7.42 A 0.344 0.24 ND 747 X33031-23-6 25 7.12 C 0.642 ---- ND 0.06

The final resin had a Brookfield viscosity of 116 cps. Scale-up 5: 350g 13.5% E7219 (unpasteurized), 4.38g Alcalase, 87.5g-23, 3.06g Nutrient Solution sample time (hours) (pH 7.50)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.63 ---- ---- 6.88 ---- ---- X33031-25-1 1 7.37-7.64 B 0.175 0.67 499 426 X33031-25-2 3 7.47-7.64 B 0.248 0.39 384 527 X33031-25-3 7 7.35-7.65 B 0.378 0.71 48 631 X33031-25-4 twenty four 7.12 FG 0.743 ---- ND 0.06

The final resin is a soft gel (dispersible). Scale-up 6: 350g 13.5% E7219 (unpasteurized), 4.38g Alcalase, 118g-25, 3.06g Nutrient Solution sample time (hours) (pH 7.3)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.41 ---- ---- 6.60 ---- ---- X33031-27-1 1 7.34-7.51 BC 0.234 0.43 466 438 X33031-27-2 7 7.13-7.33 BC 0.342 0.42 ND 766 X33031-27-3 19 6.91 BC 0.542 ---- ND 7.1

The final resin had a Brookfield viscosity of 87 cps. Scale-up 7: 350g 13.5% E7219 (unpasteurized), 4.38g Alcalase, 87.5g-27, 3.06g Nutrient Solution sample time (hours) (pH 7.35)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.44 ---- ---- 7.27 ---- ---- X33031-29-1 1 7.35-7.54 BC 0.146 0.52 676 548 X33031-29-2 4 7.30-7.65 B 0.213 0.70 319 959 X33031-29-3 8 7.37 AB 0.301 0.00 ND 1074 X33031-29-4 11 7.29-7.42 A 0.354 0.26 ND 774 X33031-29-5 1415 7.27-7.437.33 ----B 0.402---- 0.30 ND 442 X33031-29-6 twenty three 7.13 C 0.645 ---- ND 0.13 acid test <0.10 2.4

The final resin had a Brookfield viscosity of 77 cps. Scale up 8: 350g 13.5% E7219 (unpasteurized), 4.38g Alcalase, 87.5g-29, 3.06g nutrient solution sample time (hours) (pH 7.30)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.44 ---- ---- 7.17 ---- ---- X33031-31-1 1 7.37-7.46 BC 0.173 0.27 652 504 X33031-31-2 4 7.23-7.34 B 0.264 0.28 351 754 X33031-31-3 7 7.16-7.38 AB 0.361 0.46 ND 921 X33031-31-4 10 7.20-7.42 B 0.456 0.47 ND 486 X33031-31-5 13 7.20-7.39 B 0.628 0.43 ND 4.6 X33031-31-6 twenty three 7.23 BC 0.726 ND 0.14

The final resin had a Brookfield viscosity of 65 cps. Scale-up 9: 700g 13.5% E7219 (unpasteurized), 8.76g Alcalase, 175g-31, 6.12g nutrient solution sample time (hours) (pH 7.25)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.27 ---- ---- 12.86 ---- ---- X33031-33-1 1 7.18-7.39 BC 0.191 1.02 527 402 X33031-33-2 4 7.15-7.38 B 0.271 0.99 283 672 X33031-33-3 7 7.18-7.40 AB 0.337 1.05 ND 859 X33031-33-4 10 7.24-7.38 AB 0.397 0.68 ND 619 X33031-33-5 13 7.27-7.39 B 0.474 0.53 ND 361 X33031-33-6 twenty three 7.11 BC 0.697 ND 0.12 acid test <0.10 2.9

The final resin has a Brookfield viscosity of 40 cps. Scale-up 10: 350g 13.5% E7219 (unpasteurized), 4.38g Alcalase, 87.5g-33, 3.06g Nutrient Solution sample time (hours) (pH 7.20)pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.17 ---- ---- 7.20 ---- ---- X33031-35-1 1 7.15-7.34 BC 0.188 0.48 582 462 X33031-35-2 4 7.17-7.32 B 0.293 0.43 299 624 X33031-35-3 7 7.17-7.31 AB 0.380 0.45 ND 812 X33031-35-4 10 7.16-7.28 AB 0.487 0.29 ND 487 X33031-35-5 13 7.12-7.30 B 0.647 0.32 ND 150 X33031-35-6 twenty three 7.12 AB 0.820 ND 0.22 acid test <0.10 2.7

The final resin had a Brookfield viscosity of 34 cps.

Example:

For the following examples, TS means total solids

Demi Water means softened water

DO stands for dissolved oxygen

Example 23:

Demonstration of creping aid Crepetrol® 80E with increased %TS

Apply biological dehalogenation techniques to reduce 1,3-DCP and/or 3-CPD levels

Feasibility of reducing to concentrations below 1ppm

Crepetrol(R) 80E (=A3025) crepe aid resin (26.6% TS), a tertiary amine based resin available from Hercules Incorporated (Wilmington, DE), obtained from the Voreppe plant, France.

Three sterile 250ml Erlenmeyer flasks were charged with 50ml batches of resin with increasing % TS (Table 7). Dilute the resin with sterile demineralized water.

Table 7: EPI Residue and Total Solids for Crepetrol® 80E sample Crepetrol® 80E (ml) Demiwater (ml) EPI (ppm) 1,3-DCP (ppm) 2,3-DCP (ppm) 3-CPD (ppm) TS(%) 26.6% (as is) 50 0 nd 35 nd 100 26.58 20% 37.6 12.4 nd 26 nd 75 20.0 15% 28.2 21.8 nd 20 nd 56 15.0

Before inoculation, each diluted 50 ml of resin was supplemented with 0.5 ml of nutrient solution and the pH of the solution was adjusted to pH 5.8 using 33% NaOH solution. The nutrient solution contains the following components per liter of sterile demineralized water: 33g of urea, 5g of KH ₂ PO ₄ , 5g of MgSO ₄ ·7H ₂ O and 1g of CaCl ₂ ·2H ₂ O. 1 ml of the concentrated starter culture of Arthrobacter histidinophilida (HK1 ) and Agrobacterium radioactive (HK7) was removed from the -80°C freezer and thawed in a water bath at 30°C for 1-2 minutes. Both a 50 μl aliquot of the Arthrobacter histidophagicus (HK1) suspension and a 200 μl aliquot of the radioactive Agrobacterium (HK7) suspension were used to inoculate the cultures containing the 50 ml supplemented Crepetrol® 80E dilution. 250ml sterile Erlenmeyer shake flask. After inoculation, the culture was grown at 30° C. for 48 hours in a rotary shaker incubator (250 rpm; G25 type; New Brunswick Scientific Co., Inc. New Jersey, USA). Bacterial growth in time was determined by measuring its optical density at a wavelength of 600 nm using an Ultrospec 1000 UV/Vis spectrophotometer (Pharmacia Biotech, Sweden) and a 3 ml disposable cuvette with a 1-cm path length (Table 8). The pH of the sample was adjusted to pH 3.5 with concentrated sulfuric acid and 0.1% Proxel(R) BD (available from Zeneca Biocides) was added. The EPI residues (1,3-DCP and 3-CPD) of the samples were determined by GC analysis (Table 8).

Table 8: Bacterial growth in Crepetrol(R) 80E with increased %TS _OD600 value time (hours) C80E 15%TS C80E 20%TS C80E 26.6%TS 0 0.296 0.256 0.246 16.5 0.558 0.525 0.440 19.5 0.570 0.530 0.460 25 0.575 0.545 0.475 41 0.643 0.590 0.490 48 0.735 0.600 0.505 1,3-DCP (ppm) after 42 hours <1 <1 <1 3-CPD (ppm) after 42 hours <1 <1 <1

Example 24

     Sequential Enzyme and Biological Treatment with High %TS

The efficiency of Crepetrol(R) 80E treatment with ALCALASE to initiate release of 3-CPD followed by biodehalogenation was demonstrated. Biodehalogenated products of residual polymer bound 3-CPD were determined using an acid test.

A. Treatment of 2.5L Crepetrol® 80E

A clean and sterile 2.5 L bioreactor (BioFlo3000 bioreactor, controlled by AFS-BioCommand software; New Brunswick Scientific Co., Inc. New Jersey, USA) was filled with 2.5 kg of Crepetrol obtained from Hercules Voreppe plant, France 80E resin (26.6% TS). The pH of Crepetrol® 80E was adjusted to pH 7.5 using 33% NaOH solution and the pH-PID controller (Proportional Integral Display) installed in the bioreactor. Enzyme treatment was started by adding 12.5 g of Alcalase(R) 2.5LDX (Novozymes). Enzyme treat the resin for 6 hours using the following incubation conditions:

-pH 7.5

-Temperature is controlled at 25°C

- Stirring is controlled at 600rpm

Samples (25 ml) were withdrawn after 2, 4 and 6 hours to detect epi residues (Table 9). The pH of the collected samples was adjusted to pH 3.5 with concentrated sulfuric acid and stored at 4 °C for further analysis. EPI residues (3-CPD and 1,3-DCP) were analyzed by GC.

Table 9: 3-CPD released when Crepetrol 80E was treated at 26.6% TS sample Training time 3-CPD (ppm) 1,3-DCP (ppm) Crepetrol® 80E 26.6% 0 100 35 C80E-Alc1A 2 129 37 C80E-Alc1B 4 170 39 C80E-Alc1 C 6 168 39

B. Preparation of pre-cultures of HK1 and HK7 to start biodehalogenation treatment

A single colony of Arthrobacter histidinophilus (HK1) and a single colony of Agrobacterium radioactive (HK7) (both grown separately on minimal media salt medium containing DCP/CPD) were used to inoculate 50 ml sterile Brain Sterile Erlenmeyer shaker flasks (250 ml) of Heart Infusion medium (BHI; Oxoid Ltd, Basingstoke, Hampshire, England; ready-made medium, cat. no. CM225). The two precultures were cultured separately at 30° C. for 24 hours in a temperature-controlled rotary shaker incubator (250 rpm; type G25; New Brunswick Scientific Co., Inc. New Jersey, USA). The optical density of growing HK1 and HK7 cultures was determined using an Ultrospec 1000 UV/Vis spectrophotometer (Pharmacia Biotech, Sweden) at a wavelength of 600 nm using 3 ml disposable cuvettes with a 1-cm path length.

Growth was determined by measuring the optical density at 600 nm using 20-fold (water) diluted culture samples (Table 10). These precultures were used to initiate the biodehalogenation of the enzyme-treated Crepetrol(R) 80E.

Table 10: Optical Densities of HK1 and HK7 Batch Cultures Grown at 24h BHI Cultures OD ₆₀₀ (20-fold dilution) Arthrobacter histidinophilus HK1 0.386 Nitrogen colony Agrobacterium radioactive HK7 0.455

To prepare concentrated starter cultures, precultures were concentrated by centrifugation (10000 rpm for 10 minutes at 4°C) and supplemented with 10% glycerol, then stored at -80°C.

C. Biodehalogenation of Treated Crepetrol(R) 80E.

After 6 hours of enzyme treatment (Panel A), the pH of the resin in the bioreactor was adjusted to pH 5.8 with concentrated sulfuric acid. The reactor contents were supplemented with 25 ml of nutrient solution and 0.04% PPG2000 (anti-foaming agent) (polypropylene glycol P2000 (Fluka Chemie AG, Germany)). The nutrient solution contains the following components per liter of sterile demineralized water: 33g urea, 5g KH ₂ PO ₄ , 5g MgSO ₄ ·7H ₂ O and 1g CaCl ₂ ·2H ₂ O. Biodehalogenation of the enzymatically treated resin was initiated using pre-cultures of Arthrobacter histidinophila (HK1 ) and Agrobacterium radioactive (HK7) (50 ml each; panel B). Both cultures were used simultaneously to inoculate batch fermentations in 2.5 L bioreactors. The parameters of the bioreactor control unit operated in batch mode were set as follows: pH Control at pH 5.8 (PID control by adding 25% NaOH solution) temperature Controlled at 30°C stirring speed 600rpm (the maximum speed is 800rpm, controlled by DO value) ventilation Set at 1.0vvm (2.5L/min; compressed air), the minimum DO value is set at 5% air saturation

The complete removal of epi residues from the bioreactor contents was closely monitored in time by gas chromatographic analysis (GC-XSD; Table 11). After a total culture time of 51 hours, the batch culture was terminated. The pH of the enzyme-treated and biodehalogenated resin was adjusted to pH 3.5 with concentrated sulfuric acid, and the product was supplemented with 0.2% potassium sorbate and 0.12% Proxel(R) BD. A sample of the final product was used in an acid test to determine the polymer bound 3-CPD moiety. The pH of the sample (25 ml) was adjusted to pH 1.0 with concentrated sulfuric acid, and the sample was incubated at 50°C for 24 hours. After incubation, the pH was adjusted again to pH 3.5 with 33% NaOH solution. The epi residue was determined by GC-XSD assay (Table 11).

Table 11: epi residue after sequential enzyme treatment, biological treatment and acid test deal with Processing time (hours) 1,3-DCP (ppm) 3-CPD (ppm) Crepetrol® 80E 26.6% TS 0 35 100 first processing stage 246 373939 129170168 The second biological dehalogenation stage 729.550 39ndnd 168<1<1 Acid Test of C80E Raw Materials (Control) 38 188 Acid test of products nd 29

Example 25: Combined enzyme-biological treatment with high %TS

The efficiency of the treatment by Crepetrol(R) 80E to simultaneously initiate the release of 3-CPD and the simultaneous biodehalogenation of 3-CPD was demonstrated. Biodehalogenated products of residual polymer bound 3-CPD were determined using an acid test.

A clean and sterile 2.5 L bioreactor (BioFlo3000 bioreactor, controlled by AFS-BioCommand software; New Brunswick Scientific Co., Inc. New Jersey, USA) was filled with 2.5 kg of Crepetrol obtained from Hercules Voreppe plant, France 80E resin (26.6% TS). The pH of the resin was adjusted to pH 7.5 using concentrated NaOH (33%) solution, supplemented with 25 ml of nutrient solution and 0.04% PPG2000 (antifoaming agent). The nutrient solution contains the following components per liter of sterile demineralized water: 33g of urea, 5g of KH ₂ PO ₄ , 5g of MgSO ₄ ·7H ₂ O and 1g of CaCl ₂ ·2H ₂ O. Aliquots of concentrated starter cultures of Arthrobacter histidinophilum (HK1 ) and Agrobacterium radioactive (HK7) were removed from the -80°C freezer and thawed in a water bath at 30°C for 1-2 minutes. To start enzyme treatment and biodehalogenation simultaneously, add the following enzyme/bacteria amounts to the replenished resin:

-12.5g Alcalase® 2.5L DX (Novozymes)

- 0.83ml Arthrobacter histidinophilus (HK1) starter culture

- 4.17ml radioactive Agrobacterium (HK7) starter culture

The parameter settings for the bioreactor control unit operating in batch mode, during the initial processing phase, were set as follows: pH Control at pH 7.5 (PD control by adding 25% NaOH solution) temperature Controlled at 25°C stirring speed 600rpm (the maximum speed is 800rpm, controlled by DO value) ventilation Set at 1.0vvm (2.5L/min; compressed air), the minimum DO value is set at 5% air saturation

Samples were taken after 2, 4 and 6 hours to detect epi residues (Table 12). The epi residue was determined by GC. The pH of these samples (25 ml) was adjusted to pH 3.5 with concentrated sulfuric acid and stored at 4°C for further analysis.

After 6 hours of incubation, the pH of the batch was lowered to pH 5.8 with concentrated sulfuric acid and the incubation temperature was raised to 30°C. The parameters of the bioreactor control unit operating in batch mode during the "biodehalogenation phase" were set as follows: pH Control at pH 5.8 (PID control by adding 25% NaOH solution) temperature Controlled at 30°C stirring speed 600rpm (the maximum speed is 800rpm, controlled by DO value) ventilation Set at 1.0vvm (2.5L/min; compressed air), the minimum DO value is set at 5% air saturation

The complete removal of epi residues from the bioreactor contents was closely monitored in time by gas chromatographic analysis (GC-XSD; Table 12). After a total culture time of 52 hours, the batch culture was terminated. The pH of the simultaneous enzyme-treated and biodehalogenated resin was adjusted to pH 3.5 with concentrated sulfuric acid, and the product was supplemented with 0.2% potassium sorbate and 0.12% Proxel BD. A sample of the final product was used in an acid test to determine the polymer bound 3-CPD moiety. The pH of the sample (25 ml) was adjusted to pH 1.0 with concentrated sulfuric acid, and the sample was incubated at 50°C for 24 hours. After incubation, the pH was adjusted again to pH 3.5 with 33% NaOH solution. The epi residue was determined by GC-XSD assay (Table 12).

Table 12: Epi residue after simultaneous enzyme treatment and biological treatment and acid test deal with Processing time (hours) 1,3-DCP (ppm) 3-CPD (ppm) Crepetrol® 80E 26.6% TS 0 35 100 "Enzyme" treatment conditions 246 ndndnd 141135132 "Biological dehalogenation" treatment conditions 243148 <1ndnd 2<1<1 Acid Test of C80E Raw Materials (Control) 38 188 Acid test of products nd 18

Example 26: Crepetrol® 80E creping agent

Biodehalogenation of (also known as Crepetrol® A3025)

Production and preparation results

(1) Prepare total 3225L Crepetrol A3025 (available from Hercules Incorporated, Wilmington, DE) without preservatives

(2) Clean the SU2 reactor:

· Completely fill with hot water (90°C), aerate and stir. Add caustic up to pH 11.

· Hold at 90°C for 30 minutes.

• Drain the hot/caustic reactor contents into the SU1 vessel.

(3) Clean the SU1 container:

• Completely filled with hot (90°C)/caustic water from the SU2 reactor.

• Turn on aeration and agitation in the SU1 vessel during wash/heat.

· Discharge contents after 60 minutes.

Completely fill with hot water (90°C) and drain the contents of the container (= 2nd rinse).

· Heat treatment (steam) for free-draining vessel outlets, connections and all piping.

(4) pasteurize Crepetrol A3025:

• Fill reactor SU2 with 3225L Crepetrol A3025 (26% TS) and heat the feed to 80°C.

• Turn on aeration and agitation in the SU2 reactor during pasteurization.

• Keep the raw material at 80°C for 15 minutes.

• Drain 175 L (hot) of A3025 in pasteurized SU1 container.

• Drain 2000 L (hot) of A3025 in pasteurized storage container.

• The remaining 1050 L of pasteurized A3025 was retained in the SU2 reactor until further use in SU2.

• Turn off aeration and agitation.

(5) Only pasteurized water (15 minutes, 90° C.) was used for the dilution step of SU1.

(6) Add appropriate amount of K4 nutrient in dry form (see below for the amount of nutrient).

(7) Prepare 0.5 L sterile glycerol solution (161 g glycerol/500 ml; sterilize at 121° C. for 15 minutes).

Scale-up 1 (SU1): Preparation of resin inoculum

(1) Clean and pasteurize SU1 container (see above).

(2) Use 50% pasteurized and diluted raw material for SU1 container:

The SU1 container contained 175 L of pasteurized Crepetrol A3025 (26% TS) and

175 L of pasteurized (15 min, 90°C) water.

(3) Start stirring

(4) Adjust the pH in SU1 to pH 5.8±0.2 with 30% NaOH.

(5) Start to inflate the reactor (0.5vvm)

(6) Adjust the temperature in SU1 and keep it at 30±1°C.

(7) Add K4 nutrients in dry form (by rinsing the container):

· Components Concentration (g/L) 350L volume

Urea 0.33 115.5g

KH ₂ PO ₄ 0.10 35.0g

(8) Add 0.5 L (161 g/500 ml) sterile glycerol solution (= 460 ppm final concentration).

(9) Inoculate SU1 with HK1 and HK7 starter (inoculum density used: HK1 1:3500 and HK7 1:700, inoculum:resin ratio). [See Example 24 for the preparation of a concentrated starter culture]

(10) Sample determination:

- OD ₆₀₀ every 2 hours

- DCP/CPD value at the end of SU1

(11) Incubate at 30±1°C and pH 5.8±0.2 for 16-24 hours (increase pH for correction if necessary).

(12) when

-OD ₆₀₀ >0.5 or when OD ₆₀₀ value starts to stabilize

Transfer to SU2

Scale up 2

(1) Clean and pasteurize SU2 container (see above).

(2) Use 1050L pasteurized Crepetrol A3025 raw material.

(3) Start stirring

(4) Adjust the pH in SU2 to pH 5.8±0.2 with 30% sodium hydroxide.

(5) Start to inflate the reactor (0.5vvm)

(6) Adjust and maintain the temperature in SU2 at 30±1°C.

(7) Add K4 nutrients in dry form (via a clean container):

· Components Concentration (g/L) 1050L volume

Urea 0.33 346.5g

KH ₂ PO ₄ 0.10 105.0g

(8) Inoculate SU2 (inoculum density 25%) with 350 L of SU1 culture by gravity using cleaned and pasteurized connections/tubing (see above).

(10) Sample determination:

- OD ₆₀₀ every 2 hours

- DCP/CPD value at the end of SU2

(10) Incubate at 30±1°C and pH 5.8±0.2 for 16-24 hours (increase the pH for correction if necessary).

(11) when

-DCP/CPD value <5ppm or when the dissolved oxygen level increases

-Cultivation time > 24 hours

Start SU3.

Scale up 3

(1) "Pasteurized raw materials" in storage containers:

· Heat treatment (with steam) of all equipment used to mix raw materials.

· Adjust the pH to pH 5.8 ± 0.2 with 30% sodium hydroxide.

(2) Drain the storage container by gravity into the SU2 reactor (cleaned/pasteurized connections/piping) (see above).

(3) Increase the inflation volume (0.5vvm) according to the increased volume.

(4) Control the stirring and temperature (30±1°C) at the set value.

(5) Add K4 nutrients in dry form (by washing the container) for increasing the volume of 2000L:

·Components Concentration (g/L) 2000L volume

Urea 0.33 660g

KH ₂ PO ₄ 0.10 200g

(6) Sample determination:

- OD ₆₀₀ every 2 hours

- DCP/CPD value every 4 hours

- Samples for acid test at the SU3 end.

(7) Incubate at 30±1°C and pH 5.8±0.2 for 16-24 hours (add pH correction if necessary).

(8) When [DCP]+[CPD]<5ppm, the biological dehalogenation in SU3 ends.

(9) The product is completed:

Adjust the pH to pH 3.0±0.2 with concentrated sulfuric acid

Add 2000ppm (0.2%) potassium sorbate

Drain the final product through a 50-100-μm filter into a new tote.

Result: EPI-residue analysis

Table 13: Results of determination of epi-residue by GC-FID sample EPI[ppm] 1,3-DCP [ppm] 2,3-DCP [ppm] 3-CPD[ppm] Crepetrol 80E raw material ND 42 ND 131 This example ND ND ND ND Crepetrol 80E raw material after acid test ND 38 ND 227 After the acid test, this embodiment ND ND ND 56

ND = not detected

Detection limit:

EPI[ppm]: 10

1,3-DCP[ppm]: 10

2,3-DCP[ppm]: 10

3-CPD[ppm]: 10

Example 27: Biodehalogenation of Kymene® SLX2 with increased %TS

A clean and sterile 500 mL flask was filled with 380 g of Kymene(R) SLX2 (25.3% TS) obtained from Hercules Zwijndrechtplant, The Netherlands. The pH of the resin was adjusted to pH 5.8 by gradually adding (while vigorously mixing) 8.3 g of 33% NaOH solution. A series of sterilized 250ml Erlenmeyer flasks contained 50ml batches of resin with increasing %TS. Dilution of the resin was performed with sterile demineralized water (Table 14).

Table 14: Kymene® SLX2 Dilution Ranges Sample (%TS) Kymene® SLX2 (ml) Demineralized water (ml) 8 15.8 34.2 10 19.8 30.2 12 23.7 26.3 14 27.7 22.3 16 31.6 18.4 18 35.6 14.4 20 39.5 10.5 25.3 50 0

Prior to inoculation, each diluted resin sample was supplemented with 0.5 ml of filter-sterile nutrient solution. The nutrient solution contains the following components per liter of sterile demineralized water: 33g of urea, 5g of KH ₂ PO ₄ , 5g of MgSO ₄ ·7H ₂ O and 1g of CaCl ₂ ·2H ₂ O. A 1 ml sample of the concentrated starter culture of Arthrobacter histidophagae (HK1 ) and Agrobacterium radioactive (HK7) was removed from the -80°C freezer and thawed in a water bath at 30°C for 1-2 minutes. Both a 20 μl aliquot of the Arthrobacter histidinophilus (HK1) suspension and a 100 μl aliquot of the radioactive Agrobacterium (HK7) suspension were used to inoculate the 50 ml supplemented Kymene(R) SLX2 dilution. After inoculation, the culture was grown at 30° C. for 22 hours in a rotary shaker incubator (250 rpm; G25 type; New Brunswick Scientific Co., Inc. New Jersey, USA). Bacterial growth in time was determined by measuring its optical density at a wavelength of 600 nm using an Ultrospec 1000 UV/Vis spectrophotometer (Pharmacia Biotech, Sweden) and a 3 ml disposable cuvette with a 1-cm path length (Table 8). The pH of the samples was adjusted to pH 2.8 with concentrated sulfuric acid and 0.1% Proxel BD was added. The samples were determined for EPI residues (3-CPD and 1,3-DCP) by GC analysis (Table 15).

Table 15: Bacterial growth in Kymene(R) SLX2 with increased %TS Sample (%TS) _OD600nm t = 22 hours culture "Viscosity" Appearance 0 hours 17 hours 22 hours 3-CPD (ppm) 3-CPD (ppm) 8 0.141 0.655 0.654 <10 <10 - 10 0.126 0.742 0.717 <10 <10 - 12 0.122 0.797 0.762 <10 <10 - 14 0.114 0.705 0.769 <10 32 - 16 0.107 0.609 0.623 <10 294 + 18 0.094 0.556 0.560 <10 407 ++ 20 0.096 0.486 0.541 <10 492 ++ 25.3 0.095 0.139 0.130 gelled gelled +++

-: Non-stick, similar to raw material.

+: Viscous, the viscosity increased compared with the raw material.

++: Very viscous, with a sharp increase in viscosity compared to the raw material.

+++: gelling resin.

Detection limit: 10ppm 1,3-DCP

      10ppm 3-CPD.

Example 28: Kymene® E7220 with increased %TS

Biological dehalogenation (acid treated material)

A clean and sterile 500 mL flask was charged with 300 g of Kymene(R) E7220 (22.5% TS) obtained from Hercules Voreppeplant, France. The pH of the resin was adjusted to pH 7.0 by gradually adding (while vigorously mixing) 15.3 g of 33% NaOH solution. A series of sterilized 250ml Erlenmeyer flasks contained 50ml batches of resin with increasing %TS. Dilution of the resin was performed with sterile demineralized water (Table 16).

Table 16: Kymene® E7220 Dilution Range Sample (%TS) Kymene® E7220 (ml) Demineralized water (ml) 8 17.8 32.2 10 22.2 27.8 12 26.7 23.3 14 31.1 18.9 16 35.6 14.4 18 40.0 10.0 20 44.4 5.6 22.5 50 0

Prior to inoculation, each diluted resin sample was supplemented with 0.5 ml of filter-sterilized nutrient solution. The nutrient solution contains the following components per liter of sterile demineralized water: 33g urea, 5g KH ₂ PO ₄ , 5g MgSO ₄ ·7H ₂ O and 1g CaCl ₂ ·2H ₂ O. A 1 ml sample of the concentrated starter culture of Arthrobacter histidophagae (HK1 ) and Agrobacterium radioactive (HK7) was removed from the -80°C freezer and thawed in a water bath at 30°C for 1-2 minutes. Both a 20 μl aliquot of the Arthrobacter histidinophilus (HK1) suspension and a 100 μl aliquot of the radioactive Agrobacterium (HK7) suspension were used to inoculate the 50 ml supplemented Kymene(R) E7220 dilution. After inoculation (starting OD ₆₀₀ =0.208), the culture was grown at 30° C. for 91 hours in a rotary shaker incubator (250 rpm; G25 type; New Brunswick Scientific Co., Inc. New Jersey, USA). Bacterial growth in time was determined by measuring its optical density at a wavelength of 600 nm using an Ultrospec 1000 UV/Vis spectrophotometer (Pharmacia Biotech, Sweden) and a 3 ml disposable cuvette with a 1-cm path length (Table 17). The pH of the samples was adjusted to pH 2.8 with concentrated sulfuric acid and 0.1% Proxel BD was added. The samples were determined for EPI residues (3-CPD and 1,3-DCP) by GC analysis (Table 17).

Table 17: Bacterial growth in Kymene® E7220 with increased %TS Sample (%TS) _OD600nm t = 91 hours culture "Viscosity" Appearance 19 hours 27 hours 91 hours 3-CPD (ppm) 1,3-DCP (ppm) 8 1.103 1.073 nd <10 <10 - 10 1.190 1.168 nd <10 <10 - 12 1.145 1.205 nd <10 <10 - 14 0.870 1.237 nd <10 <10 - 16 0.500 1.197 1.025 <10 <10 - 18 0.276 0.647 1.095 <10 <10 - 20 0.195 0.290 1.176 <10 <10 - 25.3 0.139 0.140 1.067 <10 49 -

nd: not detected

-: Non-stick, similar to raw material.

+: Viscous, the viscosity increased compared with the raw material.

++: Very viscous, with a sharp increase in viscosity compared to the raw material.

+++: gelled resin.

Detection limit: 10ppm 1,3-DCP

      10ppm 3-CPD.

Example 29: Kymene® 736 in 15-20% TS in 50ml

Biological dehalogenation of (polyamine/azetidinium-based resins)

Kymene 736 (Crepetrol 73) crepe aid resin (39.6% TS), a polyamine/azetidinium base resin available from Hercules Incorporated (Wilmington, DE), obtained from the Voreppe plant, France.

A clean and sterile 250 mL flask was charged with 100 g of Kymene® 736 (39.6% TS) and the pH of the resin was adjusted to pH 7.0 by gradually adding (while vigorously mixing) 33% NaOH solution. Two sterile 250ml Erlenmeyer flasks were filled with 50ml batches of resin either diluted to 15% or diluted to 20% TS. Resin dilutions were performed using sterile demineralized water (Table 18).

Table 18: Kymene® 736 dilution range Sample (%TS) Kymene® 736 (ml) Demineralized water (ml) 15 19.0 31.0 20 25.2 24.8

Prior to inoculation, each diluted resin sample was supplemented with 0.5 ml of filter-sterilized nutrient solution. The nutrient solution contains the following components per liter of sterile demineralized water: 33g urea, 5g KH ₂ PO ₄ , 5g MgSO ₄ ·7H ₂ O and 1g CaCl ₂ ·2H ₂ O. A 1 ml sample of the concentrated starter culture of Arthrobacter histidophagae (HK1 ) and Agrobacterium radioactive (HK7) was removed from the -80°C freezer and thawed in a water bath at 30°C for 1-2 minutes. Both a 50 μl aliquot of the Arthrobacter histidophagicus (HK1) suspension and a 200 μl aliquot of the radioactive Agrobacterium (HK7) suspension were used to inoculate the 50 ml supplemented Kymene(R) 736 dilution. After inoculation, the culture was grown at 30° C. for 43 hours in a rotary shaker incubator (250 rpm; G25 type; New Brunswick Scientific Co., Inc. New Jersey, USA). Bacterial growth in time was determined by measuring its optical density at a wavelength of 600 nm using an Ultrospec 1000 UV/Vis spectrophotometer (PharmaciaBiotech, Sweden) and a 3 ml disposable cuvette with a 1-cm path length (Table 19). The pH of the samples was adjusted to pH 2.8 with concentrated sulfuric acid and 0.1% Proxel BD was added.

Table 19: Bacterial growth in Kymene(R) 736 with increased %TS _OD600nm time (hours) 15%TS 20%TS 0 0.441 0.378 20 0.935 0.419 twenty three 1.081 0.430 41 0.997 0.910 43 nd 0.899

nd: not detected

Example 30

Biodehalogenation of Kymene® 736 at 20% TS in a 2L batch of Kymene® 736 (Crepetrol® 73) creping aid resin (39.6% TS as is)

The crepe aid resin is a polyamine/azetidinium based resin available from Hercules Incorporated (Wilmington, DE) from the Voreppe plant, France.

A clean and sterile 2.5 L bioreactor (BioFlo3000 bioreactor, controlled by AFS-BioCommand software; New Brunswick Scientific Co., Inc. New Jersey, USA) was filled with 1010 ml of Kymene® 736 resin (39.6% TS) and 990ml sterile demineralized water. The pH of the diluted (20% TS) resin was adjusted to pH 7.0 using concentrated NaOH solution (33%), supplemented with 20 ml of nutrient solution and 0.0025% PPG2000 (antifoam). The nutrient solution contains the following components per liter of sterile demineralized water: 33g of urea, 5g of KH ₂ PO ₄ , 5g of MgSO ₄ ·7H ₂ O and 1g of CaCl ₂ ·2H ₂ O. Aliquots of concentrated starter cultures of Arthrobacter histidinophilum (HK1 ) and Agrobacterium radioactive (HK7) were removed from the -80°C freezer and thawed in a water bath at 30°C for 1-2 minutes. A 2 ml sample of Arthrobacter histidinophilum (HK1 ) suspension and an 8 ml sample of Agrobacterium radioactive (HK7) suspension were used to simultaneously inoculate the contents of a 2.5 L bioreactor. The parameters of the bioreactor control unit operated in batch mode were set as follows: pH Control at pH 7.0 (PID control by adding 25% NaOH solution) temperature Controlled at 30°C stirring speed 600rpm (the maximum speed is 800rpm, controlled by DO value) ventilation Set at 1.0vvm (2.5L/min; compressed air), the minimum DO value is set at 5% air saturation

The complete removal of epi residues from the bioreactor contents was monitored in time by gas chromatographic analysis (GC-XSD; Table 20). Batch culture was terminated after 48 hours of total culture time. The pH of the biodehalogenated resin was adjusted to pH 2.8 with concentrated sulfuric acid and the resin was supplemented with 0.02% potassium sorbate and 0.12% Proxel BD.

Table 20: Epi Residue and Bacterial Growth of Kymene® 736 at 20% TS time (hours) _OD600nm 1,3-DCP (ppm) 3-CPD (ppm) 0 0.412 - - 5 0.422 - - twenty two 0.572 - - 27 0.697 - - 30 0.818 <1 18 46 0.840 <1 <1

-:not detected

Example 31 - Alcalase treatment of tertiary amino resins

The following procedure was used to promote 3-MCPD formation by hydrolysis of polymer-linked chlorohydrin species in Crepetrol® A3025 (Hercules Incorporated, Wilmington, DE).

A 191.88 g sample of Crepetrol(R) A3025 (with no added preservatives) was placed in a 250 glass flask (equipped with a plastic-tight screw cap). Sample weight was determined with a Mettler Laboratory digital scale with an accuracy of ±0.005 g.

The total solids concentration of Crepetrol(R) A3025 was determined in a separate test measuring weight loss after 15 minutes at 150°C. The total solids concentration of the sample was found to be 26.9%.

Its pH was carefully adjusted to 7.00 with 10% w/w NaOH solution (12.52 g in total) while stirring with a magnetic stirrer, using a Mettler pH electrode equipped with an InLab electrode (combined electrode, internal standard ARGENTHAL with Ag ion trapping). - meter (MP 220) to monitor its pH. Calibrate the pH meter within the 7.00-10.00 pH range prior to its pH adjustment.

The samples were placed in an ice melting bath (0°C).

0.9 g of Alcalase(R) 2.5L DX (obtained from Novozymes) was added to the Crepetrol(R) A3025 sample.

The flask was then placed in a horizontal shaking (200 strokes/min) thermal stabilization bath at 25°C and stirred for 14 hours.

A sample was removed after 14 hours and its pH was adjusted to 3.5 with concentrated sulfuric acid.

A sample of the initial feed (Crepetrol(R) A3025) and a sample of the feed after the above treatment were analyzed using gas chromatographic techniques to determine the 3-monochloropropanediol content therein.

The amount of 3-monochloropropanediol produced during the treatment was calculated as the difference between the 3-monochloropropanediol concentration of the sample after treatment and that of the original sample Crepetrol(R) A3025.

The viscosity reduction of the final samples was measured at 25°C using an Ubbelohde capillary. A 2% 1 N ammonium chloride solution was prepared and the time to flow through the capillary was measured. The time for the ammonium chloride solution to flow was also measured. Calculate the reduced viscosity according to the following formula:

RSV[dl/g]={(time _sample [sec]/time _solvent [sec])-1}/concentration _sample [g/100ml]

The results of the CPD release are as follows:

Released 3-MCPD [ppm] = concentration _{after treatment} [ppm] - _initial concentration [ppm] = 182-101.9 = 80.1ppm

On the following page is reported a table showing the results of a series of experiments carried out with this procedure varying the conditions of enzyme dosage, pH, total solids, temperature and treatment time.

The example given above corresponds to the number 31-4 of Table 21.

Table 21 #Standard(sample tag) Enzyme g Alcalase enzyme %wt TS% TS actual calculation temperature °C pH time hours 3-MCPD release ratio δ Viscosity% δ viscosity cP RSV δRSV absolute value 31-2 0.9 0.45 15 15.0 25 7 6 0.548 -5.8 -1.6 0.265 -0.001 31-3 0.45 0.225 26 26.0 25 7 6 0.360 -1.6 -1.2 0.265 -0.001 31-9 0.47 0.235 15 15.0 25 8 6 0.486 -11.1 -3.1 0.266 0 31-12 0.9 0.45 26 26.0 25 8 6 0.964 9.2 6.6 0.274 0.008 31-21 0.675 0.3375 20.5 20.5 25 7.5 10 0.729 -16.8 -7.6 0.261 -0.005 31-1 0.45 0.225 15 15.0 25 7 14 0.568 3.9 1.1 0.265 -0.001 31-11 0.45 0.2116 26 24.4 25 8 14 0.852 49.4 31.4 0.354 0.088 31-4 0.9 0.4384 26 25.3 25 7 14 0.974 -0.5 -0.4 0.266 0 31-10 0.9 0.45 15 15.0 25 8 14 0.938 -4.7 -1.3 0.27 0.004 31-25 0.675 0.3372 20.5 20.5 32.5 7.5 2 0.325 -5.4 -2.4 0.264 -0.002 31-27 0.73 0.365 20.5 20.5 32.5 7.5 10 0.639 -7.5 -3.4 0.265 -0.001 31-29 0.675 0.3375 20.5 20.5 32.5 7.5 10 0.519 -16.8 -7.6 0.267 0.001 31-23 0.675 0.3375 20.5 20.5 32.5 6.5 10 0.218 -14.8 -6.7 0.267 0.001 31-20 0.675 0.329 26 25.3 32.5 7.5 10 0.645 -1.6 -1.1 0.28 0.014 31-24 0.675 0.3375 20.5 20.5 32.5 8.5 10 0.579 81.0 36.5 0.453 0.187 31-30 0.675 0.3374 20.5 20.5 32.5 7.5 10 0.639 -8.8 -4.0 0.266 0 31-28 0.675 0.3375 20.5 20.5 32.5 7.5 10 0.444 -9.5 -4.3 0.27 0.004 31-19 0.675 0.3375 9.5 9.5 32.5 7.5 10 0.544 -58.6 -10.2 0.27 0.004 31-18 1.13 0.5649 20.5 20.5 32.5 7.5 10 0.910 -6.2 -2.8 0.262 -0.004 31-17 0.21 0.105 20.5 20.5 32.5 7.5 10 0.203 13.8 6.2 0.272 0.006 31-26 0.675 0.3375 20.5 20.5 32.5 7.5 18 0.789 -5.5 -2.5 0.27 0.004 31-15 0.45 0.225 26 26.0 40 8 6 0.017 440.8 319.8 0.685 0.419 31-5 0.45 0.225 15 15.0 40 7 6 0.383 -14.3 -4.0 0.255 -0.011 31-8 0.9 0.45 26 26.0 40 7 6 0.609 4.2 3.0 0.277 0.011 31-14 0.9 0.45 15 15.0 40 8 6 0.814 14.2 4.0 0.319 0.053 31-6 0.9 0.45 15 15.0 40 7 14 0.938 -12.2 -3.4 0.268 0.002 31-7 0.45 0.225 26 26.0 40 7 14 0.680 24.0 17.4 0.31 0.044 31-13 0.45 0.225 15 15.0 40 8 14 0.650 93.8 26.3 0.53 0.264 31-16 0.9 0.45 26 26.0 40 8 14 na gelled gelled 1 0.734 31-22 0.675 0.3375 20.5 20.5 47.5 7.5 10 0.189 12.5 5.6 0.306 0.04

According to the reported results, the optimal conditions for the enzyme treatment of this resin are obtained through statistical calculation:

Enzyme Concentration [%W]: 0.45

TS polymer [%]: 22.17

                                                           

pH 7.94

Time [hours] 10.43

This treatment resulted in a high release of 3-MCPD (about 95%) without an increase in the final viscosity. (Higher final viscosity is an issue for product stability, especially if the resin requires additional handling).

Depending on the detailed statistical model, other conditions can be selected when desired to obtain a similar end result. The following example conditions are desired where lower amounts of enzyme are used and about 90% of the 3-MCPD is finally released and the viscosity increase is not significant.

Enzyme Concentration [%w]: 0.25

TS polymer [%]: 22.4

                                                                     

pH 8.00

Time [hours] 14.00

      Example 32: Results of Adhesion Tests

The results of the adhesion assay for a selected number of enzyme-treated samples (extracted from the series reported in the table above) are reported in the following graphs. Significant hydrolysis of the polymer (and thus reduction in average MW) can result in a significant loss of peel strength, so we wanted to check whether any significant reduction in adhesion could be detected.

Peel testing was performed by soaking a strip of fabric in a 2% solids solution of creping aid and then curing the strip in contact with a standard metal plate (mild steel) at 92°C for 7.5 minutes. The average force to peel off the strip from the metal plate was determined using a Zwick 005 universal testing machine.

The results are plotted against the change in viscosity observed after enzyme treatment. It can be clearly seen that the sample adhesion force is randomly distributed (oscillates due to experimental variation), independent of the observed viscosity variation.

Also these values are distributed around the typical values (0.75-0.8 N/cm) for untreated material.

These results show that enzyme treatment did not cause any measurable decrease in polymer adhesion strength.

Table 22 standard# Peel strengthN/cm δRSV 31-4 0.7 0 31-10 0.65 0.004 31-11 0.71 0.088 31-12 0.75 0.008 31-21 0.72 -0.005 31-18 0.81 -0.004 31-24 0.82 0.187 29 0.74 0.001 5 0.76 -0.011 6 0.93 0.002 13 0.69 0.264 14 0.77 0.053 15 0.74 0.419

Example 33: Biodehalogenation of Crepetrol® 870

(See Table 23 for data and details)

A portion of Crepetrol(R) 870 (available from Hercules Incorporated, Wilmington, DE; Voreppe, France plant) without biocide was diluted to 18.9% total solids with deionized water. The diluted resin has a Brookfield viscosity of 53 cps.

Pasteurization: A 2-L round bottom flask was equipped with a condenser, temperature controlled circulating bath and mechanical stirrer. 1780 g of 18.9% resin was added to the flask. The resin had a pH of 4.6 and was heated from 25°C to 85°C in 1 hour. The resin was held at 85°C for 20 minutes and then cooled to 25°C over 45 minutes. Pasteurized resin is stored in sterile containers.

Biodehalogenation: Preparation of resin inoculum [Scale-up 1 (SU1)]: A 250-mL round bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, air sparge tube, and mechanical stirrer. A portion of the pasteurized resin was diluted to 10% with sterile deionized water. 198 g of this 10% resin and 2.0 g of 5 mM sterile glycerol in water were added to the flask. Use 3.18 g of 30% sodium hydroxide aqueous solution to raise its pH to 5.8, then add 68 microliters of HK1 concentrated starter culture (1:3000, HK1:resin) [see Example for the preparation of concentrated starter culture 24] and add 1.75g nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 17 hours, the resulting resin was used as an inoculum for SU2.

Scale up 2 (SU2)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized 18.9% resin was added to the flask. Its pH was raised to 5.8 with 4.52 g of 30% aqueous sodium hydroxide solution, and then 50.0 g of SU1 resin inoculum (25% inoculum rate) and 1.31 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU3.

Scale up 3 (SU3)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized 18.9% resin was added to the flask. Its pH was raised to 5.8 with 4.45 g of 30% aqueous sodium hydroxide solution, and then 50.0 g of SU2 resin inoculum (25% inoculum rate) and 1.31 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 14.5 hours, the resulting resin was used as an inoculum for SU4. The remaining resin not used for the inoculum is thrown away, but can be used to obtain the final product.

Scale up 4 (SU4):

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized 18.9% resin was added to the flask. Its pH was raised to 5.8 with 8.94 g of 30% aqueous sodium hydroxide solution, and then 100.0 g of SU3 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU5. The remaining resin not used for the inoculum is thrown away, but can be used to obtain the final product.

Scale up 5 (SU5):

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized 18.9% resin was added to the flask. Its pH was raised to 5.8 with 8.96 g of 30% aqueous sodium hydroxide solution, and then 100.0 g of SU4 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 15.5 hours, the resulting resin was used as an inoculum for SU6. The remaining resin not used for the inoculum was converted to the final product by lowering its pH to 4.7 with 85% phosphoric acid and adding 2000 ppm potassium sorbate as biocide.

Scaled up 6 (SU6):

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized 18.9% resin was added to the flask. Its pH was raised to 5.8 with 8.84 g of 30% aqueous sodium hydroxide solution, and then 100.0 g of SU5 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was converted to the final product by lowering its pH to 4.7 with 7.20 g of 85% phosphoric acid and adding 2000 ppm potassium sorbate (7.11 mL of 10 wt% potassium sorbate in water) as a biocide.

See Table 23 for the results of the monitoring process.

Table 23: Acid test of Crepetrol 870 sample DCP (ppm) CPD(ppm) X33047-19A Voreppe before pasteurization ND 47 X33047-19A after acid test ND 71 X33047-39 Voreppe after pasteurization ND 43 X33047-39 after acid test ND 69 Scale up 1: 198g 10% Crepetrol 870, 2g 0.5M glycerol, 68 μl HK1, 1.75g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.81 ---- 3.18 ND twenty three X33047-41-1 1 5.81 0.020 ---- ND twenty four X33047-41-2 17 5.82 0.473 ---- 0.26 0.08 Scale up 2: 150g 18.87% Crepetrol 870, 50.0g-41, 1.31g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.82 ---- 4.52 ND 32 X33047-43-1 1 5.82 0.126 ---- ND 28 X33047-43-2 4 5.83 0.159 ---- 1.0 1.12 X33047-43-3 8 5.82 0.168 ---- 0.57 0.50 Scale up 3: 150g 18.87% Crepetrol 870, 50.0g-43, 1.31g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.81 ---- 4.45 ND 32 X33047-45-1 0.117 5.81 0.048 ---- ND 29 X33047-45-2 14.5 5.81 0.093 ---- 0.56 0.49 Scale up 4: 300g 18.87% Crepetrol 870, 100.0g-45, 2.62g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.82 ---- 8.94 ND 32 X33047-47-1 0.25 5.82 0.029 ---- ND 30 X33047-47-2 4.25 5.82 0.067 ---- 0.58 0.30 X33047-47-3 8 5.81 0.065 ---- 0.59 0.72 Scale up 5: 300g 18.87% Crepetrol 870, 100.0g-47, 2.62g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.81 ---- 8.96 ND 32 X33047-49-1 0.083 5.81 0.053 ---- ND 33 X33047-49-2 15.5 5.80 0.049 ---- 0.6 0.12 X33047-49 acid test ND 29 Scale up 6: 300g 18.87% Crepetrol 870, 100.0g-49, 2.62g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.80 ---- 8.84 ND 32 X33047-51-1 0.083 5.80 0.023 ---- ND 32 X33047-51-2 4 5.80 0.039 ---- ND 8.7 X33047-51-3 8 5.80 0.050 ---- 0.59 1.0 X33047-51 acid test ND 30

Example 34: Determination of Adhesion of Creping Agents

An apparatus for evaluating the adhesion performance of potential creping adhesives has been constructed. The device consists of a heated cast iron block mounted on the drive of the MTS measuring instrument. The platen was heated to 120°C. The paper sample is attached to the upper platen of the load cell of the measuring instrument with double-sided tape. To conduct this test, a known amount of an aqueous solution of creping adhesive of known concentration is sprayed onto a heated block. This is done with a spray gun already fitted with a volumetric spray bottle. Volumetric spray bottles allow one to accurately measure the volume of solution applied to the assay platen. Standard test conditions used were 1.2 ml of 4.0% solids in water. The pH of the solution may be close to 7.0 or may be adjusted to 7.0 prior to the assay. After spraying the resin solution onto the heated block, the drive is raised to bring the heated block into contact with the paper sample with a force of 10 kg. The drive is then lowered and the force pulls the platen away from the paper it contacts. The force thus determined is the adhesion value for the particular resin being tested. Since the applied force was not always exactly 10 kg, the adhesion force value was normalized to account for slight variations in applied force. This is obtained by multiplying the measured adhesion value by [10/(applied force (kg))]. The paper used for the assay was a 40 lb. basis weight sheet made of 50/50 hardwood/softwood bleached kraft paper.

The table below contains adhesion test and Brookfield viscosity data:

Table 24 name Viscosity (cps) Determination (Kg) (around pH) Determination (Kg) (pH 7.0) X33047-19A comparative example 53 23.4 21.7 X33047-39 comparative example 54 23.4 23.2 X33047-47 Example 45 ---- ---- X33047-49 Example 46 21.6 22.1 X33047-51 Example 49 23.7 22.2

The data in this table demonstrates that the present invention has viscosity, and adhesion tests compared to untreated over-treated resins show that biodehalogenated resins perform comparable to resins that have not been biodehalogenated.

Example 35: Alcalase-biodehalogenation of Crepetrol® 870

(See Table 25 for data and details)

A portion of Crepetrol® 870 without biocide (available from Hercules Incorporated, Wilmington, DE; Voreppe, France plant) was diluted to 18.7% total solids with deionized water. The diluted resin has a Brookfield viscosity of 53 cps.

Pasteurization: A 2-L round bottom flask equipped with a condenser, temperature controlled circulating bath and mechanical stirrer. 2942 g of 18.7% resin was added to the flask. The resin had a pH of 4.6 and was heated from 25°C to 85°C in 1.5 hours. The resin was held at 85°C for 20 minutes and then cooled to 25°C over 30 minutes. Pasteurized resin is stored in sterile containers.

Biodehalogenation: Preparation of resin inoculum [Scale-up 1 (SU1)]: A 250-mL round bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, air sparge tube, and mechanical stirrer. A portion of the pasteurized resin was diluted to 10% with sterile deionized water. 198 g of this 10% resin and 2.0 g of 5 mM sterile glycerol in water were added to the flask. Use 8.31 g of 30% sodium hydroxide aqueous solution to raise its pH to 7.2, then add 68 microliters of HK1 concentrated starter culture (1:3000, HK1:resin) [see Example for the preparation of concentrated starter culture 24], and add 1.75g nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 16 hours, the resulting resin was used as an inoculum for SU2.

Scale up 2 (SU2)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized 18.7% resin was added to the flask. Its pH was raised to 7.2 with 10.97 g of 30% aqueous sodium hydroxide solution, and then 1.02 g of Alcalase 2.5L type DX (available from Novozymes), 50.0 g of SU1 resin inoculum (25% inoculum rate) and 1.31 g's nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU3.

Scale up 3 (SU3)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized 18.7% resin was added to the flask. Its pH was raised to 7.2 with 11.42 g of 30% aqueous sodium hydroxide solution, and then 0.87 g of Alcalase 2.5L type DX (available from Novozymes), 50.0 g of SU2 resin inoculum (25% inoculum rate) and 1.31 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 14 hours, the resulting resin was used as an inoculum for SU4. The remaining resin not used for the inoculum is thrown away, but can be used to obtain the final product.

Scale up 4 (SU4)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized 18.7% resin was added to the flask. Its pH was raised to 7.2 with 22.17 g of 30% aqueous sodium hydroxide solution, and then 1.73 g of Alcalase 2.5L type DX (available from Novozymes), 100.0 g of SU3 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU5. The remaining resin not used for the inoculum was converted to the final product by lowering its pH to 4.7 with 85% phosphoric acid and adding 2000 ppm potassium sorbate as biocide.

Scale up 5 (SU5)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized 18.7% resin was added to the flask. Its pH was raised to 7.2 with 22.77 g of 30% aqueous sodium hydroxide solution, and then 1.73 g of Alcalase 2.5L type DX (available from Novozymes), 100.0 g of SU4 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 14.5 hours, the resulting resin was used as an inoculum for SU6. The remaining resin not used for the inoculum was converted to the final product by lowering its pH to 4.7 with 85% phosphoric acid and adding 2000 ppm potassium sorbate as biocide.

Scaled up 6 (SU6):

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized 18.7% resin was added to the flask. Its pH was raised to 7.2 with 23.02 g of 30% aqueous sodium hydroxide solution, and then 1.73 g of Alcalase 2.5L type DX (available from Novozymes), 100.0 g of SU5 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was converted to the final product by lowering its pH to 4.7 with 22.5 g of 85% phosphoric acid and adding 2000 ppm potassium sorbate (7.69 mL of 10 wt % potassium sorbate in water) as a biocide.

See Table 25 for the results of the monitoring treatments. Note that using this inoculum rate, the SU6 batch did not fully biodehalogenate within the 8 hr reaction time. A side-by-side experiment, with the same reaction time, using 33% inoculum in the SU4 batch and 50% inoculum in the SU5 and SU6 batches provided complete biodehalogenation in the SU6 batch (see Table 26).

Table 25: Scale up 1: 198g 10% Crepetrol 870, 2g 0.5M glycerol, 68 μl HK1, 1.75g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.20 ---- 8.31 ND twenty three X33047-62-1 0.5 7.20 0.066 ---- ND twenty three X33047-62-2 15.67 7.21 0.180 ---- ND 3.3 Scale up 2: 150g 18.73% Crepetrol 870, 50.0g-62, 1.02g Alcalase, 1.31g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.18 ---- 10.97 ND 32 X33047-64-1 1 7.16 0.180 ---- ND 34 X33047-64-2 4 7.19 0.199 ---- ND 11 X33047-64-3 8 7.18 0.253 ---- 0.41 0.48 Scale-up 3: 150g 18.73% Crepetrol 870, 50.0g-64, 0.87g Alcalase, 1.31g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.21 ---- 11.24 ND 32 X33047-66-1 0.083 7.21 0.207 ---- ND 33 X33047-66-2 13.75 7.17 0.299 ---- 0.42 0.25 Scale up 4: 300g 18.73% Crepetrol 870, 100.0g-66, 1.73g Alcalase, 2.62g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.23 ---- 22.17 ND 32 X33047-68-1 1 7.21 0.196 ---- ND 32 X33047-68-2 4 7.20 0.228 ---- ND 19 X33047-68-3 8 7.20 0.260 ---- 0.47 0.46 X33047-68 after acid test 0.37 5.9 Scale-up 5: 300g 18.73% Crepetrol 870, 100.0g-68, 1.73g Alcalase, 2.62g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.23 ---- 22.77 ND 32 X33047-70-1 0.083 7.23 0.165 ---- ND 34 X33047-70-2 14.5 7.19 0.247 ---- 0.43 0.2 X33047-70 after acid test 0.35 2.9 Scale-up 6: 300g 18.73% Crepetrol 870, 100.0g-70, 1.73g Alcalase, 2.62g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.24 ---- 23.02 ND 32 X33047-72-1 1 7.23 0.195 ---- ND 33 X33047-72-2 4 7.22 0.208 ---- ND 35 X33047-72-3 8 7.22 0.233 ---- ND 25

Table 26 Scale up 1: 198g 10% Crepetrol 870, 2g 0.5M glycerol, 68 μl HK1, 1.75g nutrient solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.20 ---- 8.20 ND twenty three X33047-63-1 0.5 7.19 0.073 ---- ND twenty three X33047-63-2 15.67 7.17 0.185 ---- ND 2.5 Scale up 2: 150g 18.73% Crepetrol 870, 50.0g-63, 1.02g Alcalase, 1.31g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.18 ---- 11.13 ND 32 X33047-65-1 1 7.17 0.181 ---- ND 32 X33047-65-2 4 7.17-7.19 0.207 0.13 ND 10 X33047-65-3 8 7.18 0.261 ---- 0.45 0.42 Scale-up 3: 150g 18.73% Crepetrol 870, 50.0g-65, 0.87g Alcalase, 1.31g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.21 ---- 11.26 ND 32 X33047-67-1 0.083 7.21 0.193 ---- ND 31 X33047-67-2 13.75 7.17 0.288 ---- 0.39 0.22 Scale-up 4: 266.7g 18.73% Crepetrol 870, 133.3g-67, 1.54g Alcalase, 2.33g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.22 ---- 20.28 ND 29 X33047-69-1 1 7.20 0.194 ---- ND twenty four X33047-69-2 4 7.20 0.234 ---- 0.45 2.7 X33047-69-3 8 7.19 0.258 ---- 0.41 0.35 Scale-up 5: 200g 18.73% Crepetrol 870, 200.0g-69, 1.16g Alcalase, 1.75g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.22 ---- 15.08 ND twenty two X33047-71-1 0.083 7.22 0.199 ---- ND 20 X33047-71-2 14.5 7.20 0.278 ---- 0.38 0.13 X33047-71 after acid test ---- 0.30 1.5 Scale-up 6: 200g 18.73% Crepetrol 870, 200.0g-71, 1.16g Alcalase, 1.75g Nutrient Solution sample time (hours) pH(30℃) _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.24 ---- 15.37 ND twenty two X33047-73-1 1 7.22 0.224 ---- ND 16 X33047-73-2 4 7.21 0.261 ---- 0.42 0.43 X33047-73-3 8 7.21 0.271 ---- 0.41 0.21 X33047-73 after acid test 0.32 3.3

Example 36: Biodehalogenation of Crepetrol® A6115

(See Table BP4 for data and details)

A portion of Crepetrol® A6115 without biocide (available from Hercules Incorporated, Wilmington, DE; Milwaukee, Wisconisin plant) was filtered through a 100 mesh screen. The resin has 15.73% total solids, pH 5.1 and a Brookfield viscosity of 86 cps.

Pasteurization: A 3-L round bottom flask was equipped with a condenser, temperature controlled circulating bath and mechanical stirrer. 2770 g of this resin was added to the flask. The resin was heated from 25°C to 85°C in 1.5 hours. The resin was held at 85°C for 20 minutes and then cooled to 25°C over 30 minutes. Pasteurized resin is stored in sterile containers.

Biodehalogenation: Preparation of resin inoculum [Scale-up 1 (SU1)]: A 250-mL round bottom flask was equipped with a condenser, pH meter, temperature-controlled circulating bath, air sparge tube, and mechanical stirrer. A portion of the pasteurized resin was diluted to 10% with sterile deionized water. 198 g of this 10% resin and 2.0 g of 5 mM sterile glycerol in water were added to the flask. Raise its pH to 6.0 with 1.04 g of 30% aqueous sodium hydroxide solution, then add 133 microliters of HK1 concentrated starter culture (1:1500, HK1:resin) [see Example for preparation of concentrated starter culture 24] and add 1.75g nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 16 hours, the resulting resin was used as an inoculum for SU2.

Scale up 2 (SU2)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized resin was added to the flask. Its pH was raised to 5.8 with 0.96 g of 30% aqueous sodium hydroxide solution, and then 50.0 g of SU1 resin inoculum (25% inoculum rate) and 1.31 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU3.

Scale up 3 (SU3)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized resin was added to the flask. Its pH was raised to 5.8 with 0.96 g of 30% aqueous sodium hydroxide solution, and then 50.0 g of SU2 resin inoculum (25% inoculum rate) and 1.31 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 14.5 hours, the resulting resin was used as an inoculum for SU4. The remaining resin not used for the inoculum is thrown away, but can be used to obtain the final product.

Scale up 4 (SU4)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized resin was added to the flask. Its pH was raised to 5.8 with 1.40 g of 30% aqueous sodium hydroxide solution, and then 100.0 g of SU3 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU5. The remaining resin not used for the inoculum is thrown away, but can be used to obtain the final product.

Scale up 5 (SU5)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized resin was added to the flask. Its pH was raised to 5.8 with 1.69 g of 30% aqueous sodium hydroxide solution, and then 100.0 g of SU4 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 15 hours, the resulting resin was used as an inoculum for SU6. The remaining resin not used for the inoculum was converted to the final product by lowering its pH to 5.3 with concentrated (96%) sulfuric acid and adding 2000 ppm potassium sorbate as a biocide.

Scale up 6 (SU6)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized resin was added to the flask. Its pH was raised to 5.8 with 1.82 g of 30% aqueous sodium hydroxide solution, and then 100.0 g of SU5 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution were added. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was converted to the final product by lowering its pH to 5.3 with 0.73 g of concentrated (96%) sulfuric acid and adding 2000 ppm of potassium sorbate (7.6 mL of 10 wt % potassium sorbate in water) as a biocide .

See Table 27 for the results of the monitoring treatments.

Table 27: Scale-up 1: 198g 10% Crepetrol A6115 (pasteurized), 2g 0.5M glycerol, 133 μl HK1 (1:1500, resin:inoculum), 1.75g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.96 ---- ---- 1.04 1.7 30 X32989-58-1 0.25 5.95 ---- 0.059 ---- ND 30 X32989-58-2 16 5.95 ---- 0.355 ---- 1.2 0.28 Scale up 2: 150g 15.7% Crepetrol A6115 (pasteurized), 50.0g-58, 1.31g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.81 ---- ---- 0.96 2.0 35 X32989-60-1 1 5.81 ---- 0.134 ---- 2.0 0.50 X32989-60-2 4 5.82 ---- 0.130 ---- 1.9 0.42 X32989-60-3 8 5.83 ---- 0.124 ---- 1.9 0.58 Scale up 3: 150g 15.7% Crepetrol A6115 (pasteurized), 50.0g-60, 1.31g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.81 ---- ---- 0.96 2.0 35 X32989-62-1 0.083 5.81 ---- 0.080 ---- ND 28 X32989-62-2 14.5 5.81 ---- 0.078 ---- 2.8 0.59 Scale-up 4: 300g 15.7% Crepetrol A6115 (pasteurized), 100.0g-62, 2.62g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.78 ---- ---- 1.40 2.0 35 X32989-64-1 1 5.77 ---- 0.064 ---- ND twenty four X32989-64-2 4 5.76 ---- 0.050 ---- 2.3 0.39 X32989-64-3 8 5.77 ---- 0.055 ---- 2.3 0.49 Scale up 5: 300g 15.7% Crepetrol A6115 (pasteurized), 100.0g-64, 2.62g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.80 ---- ---- 1.69 2.0 35 X32989-66-1 0.083 5.76 E. 0.054 ---- ND 33 X32989-66-2 15 5.76 E. 0.042 ---- 2.3 0.55 Scale up 6: 300g 15.7% Crepetrol A6115 (pasteurized), 100.0g-66, 2.62g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 5.77 ---- ---- 1.82 2.0 35 X32989-68-1 1 5.76 E. 0.036 ---- ND twenty four X32989-68-2 4 5.75 ---- 0.054 ---- 2.1 1.7 X32989-68-3 8 5.75 E. 0.039 ---- 2.2 0.41

Example 37: Alcalase-biodehalogenation of Crepetrol® A6115 wrinkling agent

(See Table BP5 for data and details)

A portion of Crepetrol(R) A6115 without biocide (available from Hercules Incorporated, Wilmington, DE; Milwaukee, Wisconisin plant) was filtered through a 100 mesh screen. The resin has 15.73% total solids, pH 5.1 and a Brookfield viscosity of 86 cps.

Pasteurization: A 3-L round bottom flask was equipped with a condenser, temperature controlled circulating bath and mechanical stirrer. 2770 g of this resin was added to the flask. The resin was heated from 25°C to 85°C in 1.5 hours. The resin was held at 85°C for 20 minutes and then cooled to 25°C over 30 minutes. Pasteurized resin is stored in sterile containers.

Biodehalogenation: preparation of resin inoculum

Scale up 1 (SU1)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. A portion of the pasteurized resin was diluted to 10% with sterile deionized water. 198 g of this 10% resin and 2.0 g of 5 mM sterile glycerol in water were added to the flask. Its pH was raised to 7.2 with 2.65 g of 30% aqueous sodium hydroxide solution, then 0.62 g of Alcalase 2.5L type DX (available from Novozymes), 133 microliters of HK1 concentrated starter culture (1:1500, HK1: resin) [see Example 24 for the preparation of concentrated starter culture] and add 1.75 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 16 hours, the resulting resin was used as an inoculum for SU2.

Scale up 2 (SU2)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized resin was added to the flask. Its pH was raised to 7.2 with 3.37 g of 30% aqueous sodium hydroxide solution, and then 0.73 g of Alcalase 2.5L type DX (available from Novozymes), 50.0 g of SU1 resin inoculum (25% inoculum rate) and 1.31 g's nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU3.

Scale up 3 (SU3)

A 250-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 150 g of the pasteurized resin was added to the flask. Its pH was raised to 7.2 with 3.02 g of 30% aqueous sodium hydroxide solution, and then 0.73 g of Alcalase 2.5L type DX (available from Novozymes), 50.0 g of SU2 resin inoculum (25% inoculum rate) and 1.31 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. _OD600 was determined by measuring its optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length of. After 14.5 hours, the resulting resin was used as an inoculum for SU4. The remaining resin not used for the inoculum is thrown away, but can be used to obtain the final product.

Scale up 4 (SU4)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized resin was added to the flask. Its pH was raised to 7.2 with 6.03 g of 30% aqueous sodium hydroxide solution, and then 1.46 g of Alcalase 2.5L type DX (available from Novozymes), 100.0 g of SU3 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was used as an inoculum for SU5. The remaining resin not used for the inoculum is thrown away, but can be used to obtain the final product.

Scale up 5 (SU5)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized resin was added to the flask. Its pH was raised to 7.2 with 6.26 g of 30% aqueous sodium hydroxide solution, then 1.46 g of Alcalse 2.5L type DX (available from Novozymes), 100.0 g SU4 resin inoculum (25% inoculum rate) and 2.62 g nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 15 hours, the resulting resin was used as an inoculum for SU6. The remaining resin not used for the inoculum was converted to the final product by lowering its pH to 5.3 with concentrated (96%) sulfuric acid and adding 2000 ppm potassium sorbate as biocide.

Scale up 6 (SU6)

A 500-mL round bottom flask was equipped with a condenser, pH meter, circulating bath for temperature control, air sparge tube, and mechanical stirrer. 300 g of the pasteurized resin was added to the flask. Its pH was raised to 7.2 with 6.02 g of 30% aqueous sodium hydroxide solution, and then 1.46 g of Alcalase 2.5L type DX (available from Novozymes), 100.0 g of SU5 resin inoculum (25% inoculum rate) and 2.62 g of nutrient solution. (The nutrient solution consisted of tap water of 8026ppm potassium dihydrogen phosphate, 27480ppm urea, 4160ppm magnesium sulfate and 840ppm calcium chloride.) Air sparging was started and the temperature was maintained at 30°C. Bacterial growth was monitored by optical density ( _OD600 ) and biodehalogenation by GC. OD ₆₀₀ is determined by measuring the optical density at a wavelength of 600 nm using a Spectronic® Genesys ^™ UV/Vis spectrophotometer (Spectronic Instruments, Incorporated, Rochester, New York, USA) and a disposable cuvette with a 1-cm path length. definite. After 8 hours, the resulting resin was converted to the final product by lowering its pH to 5.3 with 2.92 g of concentrated (96%) sulfuric acid and adding 2000 ppm of potassium sorbate (7.6 mL of 10 wt % potassium sorbate in water) as a biocide .

See Table 28 for the results of the monitoring process.

Table 28: Scale up 1: 198g 10% Crepetrol A6115 (pasteurized), 2g 0.5M glycerol, 0.62g Alcalase, 133 microliters HK1 (1:1500, resin:inoculum), 1.75g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.17 ---- ---- 2.65 1.7 30 X32989-59-1 0.25 7.16 ---- 0.058 ---- ND 32 X32989-59-2 16 7.17 ---- 0.450 ---- 0.57 0.12 Scale up 2: 150g 15.7% Crepetrol A6115, 0.73g Alcalase, 50.0g-59, 1.31g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.19 ---- ---- 3.37 2.0 35 X32989-61-1 1 7.18 ---- 0.167 ---- 1.7 3.6 X32989-61-2 4 7.19 ---- 0.180 ---- 1.5 0.27 X32989-61-3 8 7.18 ---- 0.183 ---- 1.4 0.24 Scale up 3: 150g 15.7% Crepetrol A6115, 0.73g Alcalase, 50.0g-61, 1.31g nutrient solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.17 ---- ---- 3.02 2.0 35 X32989-63-1 0.083 7.16 ---- 0.087 ---- ND 33 X32989-63-2 14.5 7.16 ---- 0.130 ---- 1.5 0.22 Scale-up 4: 300g 15.7% Crepetrol A6115, 1.46g Alcalase, 100.0g-63, 2.62g Nutrient Solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.15 ---- ---- 6.03 2.0 35 X32989-65-1 1 7.13 ---- 0.079 ---- ND 31 X32989-65-2 4 7.12 ---- 0.095 ---- 1.9 0.59 X32989-65-3 8 7.11 ---- 0.110 ---- 1.9 0.37 Scale-up 5: 300g 15.7% Crepetrol A6115, 1.46g Alcalase, 100.0g-65, 2.62g Nutrient Solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.20 ---- ---- 6.26 2.0 35 X32989-67-1 0.083 7.17 C 0.050 ---- ND 35 X32989-67-2 15 7.17 C 0.107 ---- 1.7 0.19 Scale-up 6: 300g 15.7% Crepetrol A6115, 1.46g Alcalase, 100.0g-67, 2.62g Nutrient Solution sample time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 0 7.16 ---- ---- 6.02 2.0 35 X32989-69-1 1 7.14 C 0.061 ---- 2.5 32 X32989-69-2 4 7.13 ---- 0.099 ---- 1.9 1.1 X32989-69-3 8 7.13 C 0.107 ---- 1.8 0.23

   Example 38: High solids, simultaneous enzyme-biological dehalogenation treatment

General steps

A low molecular weight terpolymer was prepared by condensing the reactants adipic acid, diethylenetriamine and acetic acid in a molar ratio of 1:0.9:0.2 at 170°C for 3 hours. The reaction product was diluted to 50% solids.

These polymers were reacted with epichlorohydrin at an epichlorohydrin:diethylenetriamine ratio of 0.82 at 40°C for 3.5 hours and water was added so that the total solids content of the reactants was 40%.

In the next step, the reaction mixture was diluted to a total solids content of 31% and heated to 68 °C for functionalization and crosslinking. After about 2 hours 10 minutes, at this temperature, the reaction was stopped at Gardner-Holdt viscosity "I/J" by the addition of 30% sulfuric acid to bring the pH after sulfuric acid addition to 4.5. The reaction product was cooled to room temperature, 1.75% phosphoric acid (weight:reactant volume) was added, and after adding phosphoric acid, its pH was adjusted to pH 2.7 using sulfuric acid. The purpose of adding phosphoric and sulfuric acid to this pH is to obtain a viscometrically stable resin.

The resins were analyzed for residual organochlorine levels and the 1,3-DCP level was found to be 816 ppm for the acetic acid containing material. These resins were tested in paper tests and found to be effective in imparting wet strength to paper as Kymene(R) SLX2. The resins were stored at 32°C for 6 weeks, during which time the resins did not gel.

Biodehalogenation (see Table 29 for data and details): Inoculum was prepared with uncapped resin (Kymene E7129) (see Scale-up 1 and Scale-up 2 of Table 29). The capped resin prepared above was diluted to 13.5% solids, its pH was raised to pH 7.2 with 30% aqueous sodium hydroxide solution, and a catalyst (Alcalase, Novozymes) for hydrolysis of CPD-forming species was added, obtained from the inoculum of scale-up 2. substances and nutrients. Without wishing to be bound by theory, it is believed that this low solids intermediate step is useful in increasing the adaptability of the microbial population to the new resin. After completion of the biodehalogenation, the next batch (scale-up 4) was started. The capped resin prepared above was diluted to 20% solids, its pH was raised to pH 7.2 with 30% aqueous sodium hydroxide solution, and a catalyst (Alcalase, Novozymes) for hydrolysis of CPD-forming species was added, obtained from the inoculum of scale-up 3 material (20% inoculum rate) and nutrient solution. The microorganisms grew rapidly as indicated by optical density ( _OD600 ) (see scale-up 4 in Table 29). Biodehalogenation was also rapid, as indicated by the rapid loss of DCP and CPD.

Table 29 Alcalase-biological dehalogenation of high solid wet strength resin Scale up 1: 200 g 8% E7219 (autoclaved), no Alcalase, 400 μl HK7, 1.75 g nutrient solution sample time time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 6:56 0 7.15 ---- ---- 2.54 ---- ---- X32966-3-1 7:58 1 7.14 ---- 0.171 ---- 213 143 X32966-3-2 10:57 4 7.13 ---- 0.162 ---- 144 181 X32966-3-3 13:58 7 7.08-7.25 ---- 0.162 0.11 62 235 X32966-3-4 17:00 10 7.21 ---- 0.184 ---- ND 274 X32966-3-5 20:10 13 7.17 ---- 0.204 ---- ND 228 X32966-3-6 5:55 twenty three 7.03 ---- 0.509 ---- ND 2.6 Scale-up 2: 350g 13.5% E7219 (pasteurized), 5.03g Alcalase, 87.5g-3, 3.06g nutrient solution sample time time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 6:40 0 7.23 ---- ---- 7.31 ---- ---- X32966-5-1 7:40 1 7.20 ---- 0.113 ---- 80 391 X32966-5-2 10:41 4 7.04-7.21 ---- 0.149 0.33 ND 415 X32966-5-3 13:30 7 7.19 ---- 0.248 ---- ND 313 X32966-5-4 16:50 10 7.11-7.27 ---- 0.383 0.31 ND 98 X32966-5-5 20:50 14 7.28 ---- 0.475 ---- ND 38 X32966-5-6 5:59 twenty three 7.17 ---- 0.573 ---- ND 0.43

The final resin had a Brookfield viscosity of 38 cps. Scale-up 3: 200g 13.5% capped resin, 2.50g Alcalase, 22.22g-5, 1.75g nutrient solution sample time time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 7:00 0 7.22 ---- ---- 5.71 ---- ---- X32966-11-1 8:01 1 7.18 ---- 0.059 ---- 270 343 X32966-11-2 11:02 4 7.05-7.23 ---- 0.092 0.28 40 621 X32966-11-3 14:01 7 7.17 ---- 0.165 ---- ND 660 X32966-11-4 17:00 10 7.15-7.31 ---- 0.277 0.15 ND 627 X32966-11-5 5:50 twenty three 7.11 ---- 0.772 ---- ND 0.4 Scale-up 4: 160g 20% capped resin, 3.00g Alcalase, 40.0g-11, 1.40g nutrient solution sample time time (hours) pH(30℃) Gardner viscosity _OD600 (absolute value) 30%NaOH(g) DCP (ppm) CPD(ppm) ---- 6:56 0 7.25 ---- ---- 6.09 ---- ---- X32966-15-1 8:00 1 7.21 AB 0.148 ---- 406 393 X32966-15-2 12:15 5.3 7.06-7.23 AA-1 0.346 0.27 283 586 X32966-15-3 17:45 10.6 7.03-7.16 AA-1 0.728 0.23 86 14 X32966-15-4 9:30 26.5 7.06 AA-1 0.962 ---- ND 0.68 X32966-15-5 8:30 49.5 6.91 AA-1 1.004 ---- ND 0.22

Claims (47)

1, a way for polyamine - epihalohydrin resin storage stable, comprising:

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one wet process comprising Strength polyamine - epihalohydrin resin composition solids content of the composition is at least 15 wt%, and contains CPD-forming substances in order to reduce the CPD containing polyamine to form - Epihalohydrin resin composition as at 50 ℃, pH about 1.0 released after 24 hours storage under CPD put dry weight of less than about 250ppm.

2 A the polyamine - epihalohydrin resin storage stable, comprising:

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one process containing a poly Amine - epihalohydrin resin composition solids content of the composition is at least 15wt%, And containing CPD-forming substance in order to reduce the CPD containing polyamine formation - epihalohydrin When the resin composition is 50 ℃, pH about 1.0 to 24 hours following the release of stored CPD Dry weight of less than about 100ppm.

3, The method as claimed in claims 1-2, wherein the reduced CPD formation comprising polyamine - epihalohydrin When the resin composition is 50 ℃, pH about 1.0 to 24 hours storage under contain less than about 5 0ppm dry weight of the CPD.

4, The method as claimed in claims 1-2, wherein said process conditions comprise about 20 ℃ -60 ℃ temperature Degrees.

5, The method as claimed in claims 1-2, wherein said process conditions comprise about 20 ℃ -40 ℃ temperature Degrees.

6 The method as claimed in claims 1-2, wherein said process conditions comprise about 30 minutes - about 96 Hours of reaction time.

7, The method as claimed in claim 6, wherein said process conditions comprise about 2 hours - about 12 hours Reaction time.

8, The method as claimed in claims 1-2, wherein said process conditions comprise about 2.5 to about 9 pH.

9, The method as claimed in claim 8, wherein said process conditions comprise about 7 - about 9 pH.

10, The method as claimed in claim 9, wherein said process conditions comprise from about 6 - about 8.5 pH.

11, The method as claimed in claims 1-2, wherein at least one enzyme reagent with a polyamine - epihalohydrin tree The ratio of fat dry basis is approximately 1:1600 - about 1:1.5.

12, The method as claimed in claim 11, wherein at least one enzyme reagent with a polyamine - epihalohydrin tree The ratio of fat dry basis is approximately 1:160 - about 1:4.

13, The method as claimed in claims 1-2, wherein at least one active agent dry enzyme with a polyamine - Table Halogenated alcohol resin dry ratio is approximately 0.04:1600 - about 0.04:1.5.

14, The method as claimed in claims 1-2, wherein the solid content of 15-50wt% active solids, The processing conditions include a temperature of from about 0 ℃ - about 35 ℃, the reaction time is about 4 - about 24 Hours, pH of about 6.9 to about 7.9, at least one enzyme reagent with a polyamine - epihalohydrin resin Dry ratio is approximately 1:20 - 1:8.

15, The method as claimed in claims 1-2, wherein said at least one reagent selected from esterase enzyme, fat Lipase, protease or mixtures thereof.

16, The method of claim 15, wherein said at least one reagent is a subtilisin enzyme The protease enzyme group.

17, The method as claimed in claims 1-2, wherein said at least one enzyme with esterase activity reagent.

18, The method as claimed in claims 1-2, wherein said agent is at least one enzyme selected from lichens Bacillus subtilis (Swiss-Prot Accession Number: P00780) or Bacillus amyloliquefaciens Coli (P00782), and slow Bacillus (P29600) microbial production.

19, The method as claimed in claims 1-2, wherein said agent is at least one enzyme ALCALASE.

19, The method as claimed in claims 1-2, wherein said agent is at least one enzyme ALCALASE....

19, The method as claimed in claims 1-2, wherein said agent is at least one enzyme ALCALASE....

22, The method as claimed in claims 1-2, wherein said resin is characterized by the presence of the following formula Functional groups:

Wherein X-is an anion.

23, The method as claimed in claims 1-2, wherein the processing polyamine - epihalohydrin resin composition CPD was reduced to obtain a resin formed at the same time, before or after the one in which at least When the resin with at least one microorganism or a microorganism isolated from said at least one Contacting at least one enzyme, and pH, and temperature in an amount such that the residual amounts of effective Connect dehalogenation of organic halogen.

24, The method as claimed in claims 1-2, wherein the processing polyamine - epihalohydrin resin composition Was reduced to obtain a resin formed while CPD, CPD-forming resin and to the At least one microorganism or a microorganism isolated from said at least one connection of at least one enzyme Touch, the amount and pH, and temperature such that the residual effective amount of an organic halogen connection Dehalogenation.

25, as claimed in claim 23 or 24, wherein said at least one microorganism, or from a At least one of said at least one enzyme isolated microorganism is a hydrogen halide lyases dehalogenation Enzymes.

26, as claimed in claim 23 or 24, wherein said at least one microorganism, or from a At least one of said at least one enzyme isolated microorganism include Arthrobacter macrophage histidinol (HK1) And radioactive Agrobacterium (HK7) at least one.

27, as claimed in claim 23 or 24, wherein said microorganism comprises at least one discharge containing Radioactive Agrobacterium (HK7) and macrophage histidinol Arthrobacter (HK1) mixing at least one of Thereof.

28, as claimed in claim 23 or 24, wherein said process conditions comprise 48 hours or more Less reaction time.

29, as claimed in claim 23 or 24, wherein said temperature is about 20 ℃ -35 ℃.

30, as claimed in claim 23 or 24, wherein said process condition comprises about 6.5 to 8.0 pH.

31, as claimed in claim 23 or 24, wherein at least one enzyme reagent with a polyamine - epihalohydrin The ratio of alcohol resin dry basis is approximately 1:1600 - about 1:1.5.

32, as claimed in claim 23 or 24, wherein said process conditions comprise reaction time To 48 hours or less, a temperature of about 20 ℃ -35 ℃, pH of about 6.5 to about 8.0, at least one Enzyme reagent with a polyamine - epihalohydrin resin dry ratio is approximately 1:1600 - about 1:1.5, and And said at least one microorganism Agrobacterium containing radioactive included (HK7) and macrophages histidinol Arthrobacter (HK1) a mixture of at least one.

33, The method as claimed in claims 1-2, wherein the processing polyamine - epihalohydrin resin composition CPD was reduced to obtain a resin formed at the same time, before or after the one in which at least When the resin is treated to reduce the epihalohydrin, epihalohydrin hydrolysis by-products and Attached to the polymer backbone of at least one organic halogen.

34, A process for preparing a paper product, comprising:

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one wet process comprising Strength polyamine - epihalohydrin resin composition solids content of the composition is at least 15 wt%, and contains CPD-forming substance and used to reduce the formation of CPD polyamine - epihalohydrin Alcohol resin is formed of paper products so that the paper products, when adding an additional amount of about 1wt% of the A resin to reduce the CPD correction, contains less than about 250ppb of CPD.

35, A process for preparing a paper product, comprising:

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one process containing a poly Amine - epihalohydrin resin composition solids content of the composition is at least 15wt%, And containing CPD-forming substance, and used to reduce the formation of CPD polyamine - epihalohydrin resin Formation of paper products, to paper products, when adding an additional amount of about 1wt% of the reduction CP D is corrected when a resin containing less than about 100ppb of CPD.

36, as claimed in claim 34 or 35, wherein the paper product, when adding an additional amount of about 1wt% Reduction of said corrected CPD when a resin containing less than about 50ppb of CPD.

37, as claimed in claim 34 or 35, wherein the solid content of 15-50wt% active Solid, the reaction temperature is about 0 ℃ - about 35 ℃, the reaction time is about 4 - about 24 hours, The reaction pH of about 6.9 to about 7.9, at least one enzyme reagent with a polyamine - epihalohydrin resin Dry ratio is approximately 1:20 - 1:8.

38, a treatment of organic halogen-containing compound and a nitrogen-polyamine - epihalohydrin resin aqueous composition Nitrogen-free organic material to reduce the amount of a halogen compound, comprising:

In the nitrogen-free organohalogen compound under conditions dehalogenation to the aqueous composition is added to the At least one microorganism, or at least one from said at least one microorganism isolated enzyme, Solids content of the composition is at least 15wt%, and containing CPD-forming substance, so that Nitrogen-free organohalogen compound reduces the amount of both the polyamine - epihalohydrin resin is substantially free from Affected.

39, The method of claim 38, wherein said solids content is about 18-35wt%.

40 A the polyamine - epihalohydrin resin storage stable, comprising:

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one wet process comprising Strength polyamine - epihalohydrin resin composition solids content of the composition is at least 15 wt%, and contains CPD-forming substances in order to reduce the CPD containing polyamine to form - Epihalohydrin resin composition as at 50 ℃, pH about 1.0 to 24 hours storage under release CPD put dry weight of less than about 250ppm, which deal with the polyamine - epihalohydrin tree To obtain a grease composition of the resin to reduce the formation of CPD same time, the shape of the CPD A synthetic resin with at least one microorganism, or from said at least one microorganism isolated from Contacting at least one enzyme, and pH, and temperature in an amount effective to make the residual amount of organic Connect halogen dehalogenation. ...

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one wet process comprising Strength polyamine - epihalohydrin resin composition solids content of the composition is at least 15 wt%, and contains CPD-forming substances in order to reduce the CPD containing polyamine to form - Epihalohydrin resin composition as at 50 ℃, pH about 1.0 to 24 hours storage under release CPD put dry weight of less than about 250ppm, which deal with the polyamine - epihalohydrin tree To obtain a grease composition of the resin to reduce the formation of CPD same time, the shape of the CPD A synthetic resin with at least one microorganism, or from said at least one microorganism isolated from Contacting at least one enzyme, and pH, and temperature in an amount effective to make the residual amount of organic Connect halogen dehalogenation. ...

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one process containing a poly Amine - wrinkled epihalohydrin resin composition solids content of the composition is at least 15wt % And contains CPD-forming substance in order to reduce the CPD containing polyamine formation - epihalohydrin Generation alcohol resin compositions when 50 ℃, pH about 1.0 after 24 hours storage under the release of CPD dry weight of less than about 100ppm, in which the processing polyamine - epihalohydrin resin Reducing composition to obtain a resin formed while CPD, CPD forming said tree Fat and at least one microorganism, or at least one microorganism isolated from the at least one of Enzyme exposure, and pH, and temperature in an amount effective to make the amount of residual organic linking Halogen dehalogenation. ...

With at least one enzyme reagent in the inhibition, reduction and removal of CPD-forming substance to obtain a gelled Storage stability and reduce the formation of CPD in the resin under conditions of at least one process containing a poly Amine - wrinkled epihalohydrin resin composition solids content of the composition is at least 15wt % And contains CPD-forming substance in order to reduce the CPD containing polyamine formation - epihalohydrin Generation alcohol resin compositions when 50 ℃, pH about 1.0 after 24 hours storage under the release of CPD dry weight of less than about 100ppm, in which the processing polyamine - epihalohydrin resin Reducing composition to obtain a resin formed while CPD, CPD forming said tree Fat and at least one microorganism, or at least one microorganism isolated from the at least one of Enzyme exposure, and pH, and temperature in an amount effective to make the amount of residual organic linking Halogen dehalogenation. ...

43, The method as claimed in claim 40-42, wherein the solid content of 4 - about 14.5wt%.

44, The method as claimed in claim 40-42, wherein said solids content of 8 - about 14.5wt%.

45, The method as claimed in claim 38-42, wherein said at least one microorganism, or from the Said at least one microorganism is at least one enzyme isolated hydrogen halide lyases dehalogenase.

46, The method as claimed in claim 38-42, wherein said at least one microorganism, or from the Said at least one microorganism comprises at least one enzyme isolated macrophages histidinol Arthrobacter (HK1) And radioactive Agrobacterium (HK7) at least one of.

47, The method as claimed in claim 38-42, wherein said microorganism comprises at least one radiation-containing Soil bacteria (HK7) and macrophage histidinol Arthrobacter (HK1) in a mixture of at least one.